Working Group #01 Consolidated Post-Conference Call Report.doc

NCDD Overview of the Digestive System Working Group (WG 1)Consolidated Pre-Conference Call Report

Chair: Richard S. Blumberg, MD, Brigham and Women’s Hospital, BostonVice Chair: Eugene B. Chang, MD, University of Chicago

This document is a collection of the revised reports that were submitted by the above-named NCDD Working Group (WG) members following their February 26, 2007, conference call.

The reports contain the members’ revised goals based on discussions during the call. This revised report will be the basis for the Working Group’s second conference call on

Friday, March 30, 20073:00 to 5:00 pm Eastern Time

List of Current ReportsPage

Development of the GI Tract, Ramesh Shivdasani, MD, PhD .....................................................................2Growth and Integrative Physiology, Hannah Carey, PhD.............................................................................7Digestion, Nicholas Davidson, MD.............................................................................................................13Nutrient Fluid Absorption/Secretion, Marshall Montrose, PhD..................................................................18Neurophysiology and Endocrinology, Chung Owyang, MD.......................................................................22Intestinal Microbiota and Digestive Health, Abigail, Salyers, PhD............................................................32Mucosal Immunology, Warren Strober, MD...............................................................................................40

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NAME: Ramesh Shivdasani, MD, PhD, Harvard Medical School; Dana Farber Cancer InstituteWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Development of the GI Tract

OVERVIEW: Many GI disorders, ranging from congenital malformations to inflammatory bowel disease, and malabsorption to metaplasia and cancer, are thought to have some basis in development of the embryonic gut. Accordingly, GI development has long been a priority for the NIH, which supported visionary research leading to improved understanding of endoderm specification, patterning, stem/progenitor cell kinetics, and crypt-villus organization. These advances permit the field to take the next vital steps and dissect the molecular basis of GI development, which holds enormous potential for pathophysiology and therapy. In the face of unprecedented scientific opportunities, the NIH is positioned to draft a bold agenda for molecular investigation of development of the gut and its affiliated organs. Although the research advances cited below are primarily in the realm of basic research, they touch on common problems in human health, especially birth defects, cancer and inflammation, and are highly relevant to emerging ideas and technologies in regenerative medicine.

1. RESEARCH ADVANCES

Research Advance #1

Elucidation of seminal pathways in development and maintenance of the mammalian gut and improved understanding of interactions between these pathways in genesis of the GI tract.

An important challenge in development and homeostasis of all tissues is to understand how a limited number of signaling pathways is able to generate enormous diversity. This question is starting to yield insights in the GI tract, where the roles of the Wnt, Notch, Hedgehog, BMP, and FGF pathways, among others, are beginning to be defined. Recent advances help delineate how these widely expressed signaling pathways act in concert to generate tissue- and organ-specific structures and functions and they establish the GI tract as an exceptional model system to study developmental mechanisms.

Selected citations:

1. Ramalho-Santos, M., Melton, D. A. and McMahon, A. P. (2000). Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127, 2763-2772.

2. van den Brink, G. R., Hardwick, J. C., Tytgat, G. N., Brink, M. A., Ten Kate, F. J., Van Deventer, S. J. and Peppelenbosch, M. P. (2001). Sonic hedgehog regulates gastric gland morphogenesis in man and mouse. Gastroenterology 121, 317-328.

3. Batlle, E., Henderson, J. T., Beghtel, H., van den Born, M. M., Sancho, E., Huls, G., Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T. et al. (2002). Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251-263.

4. Sansom, O. J., Reed, K. R., Hayes, A. J., Ireland, H., Brinkmann, H., Newton, I. P., Batlle, E., Simon-Assmann, P., Clevers, H., Nathke, I. S. et al. (2004). Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 18, 1385-1390.

5. Fre, S., Huyghe, M., Mourikis, P., Robine, S., Louvard, D. and Artavanis-Tsakonas, S. (2005). Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964-968.

Research Advance #2

Deeper understanding of the molecular underpinnings of the intestinal crypt-villus axis, its relation to intestinal carcinomas, and of information transmitted by the underlying stroma.

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One of the triumphs of recent basic research is the elucidation of the role of the Wnt signaling pathway in intestinal crypt homeostasis and in distinguishing the functions of crypt progenitors from those of differentiated villus epithelial cells. These advances relate in powerful ways the genetic basis of colorectal cancer, the second leading cause of U.S. cancer deaths, to epithelial stem cell homeostatic mechanisms. It is increasingly clear that Wnt signaling maintains proliferative capacity and lack of differentiation in crypts; its absence permits differentiation in villi; and that constitutive Wnt activity is responsible in large part for dysregulated cell proliferation in colorectal and some other GI tumors.

Selected citations:

1. van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P. et al. (2002). The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-250.

2. Haramis, A. P., Begthel, H., van den Born, M., van Es, J., Jonkheer, S., Offerhaus, G. J. and Clevers, H. (2004). De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684-1686.

3. He, X. C., Zhang, J., Tong, W. G., Tawfik, O., Ross, J., Scoville, D. H., Tian, Q., Zeng, X., He, X., Wiedemann, L. M. et al. (2004). BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet 36, 1117-1121.

4. Perreault, N., Sackett, S. D., Katz, J. P., Furth, E. E. and Kaestner, K. H. (2005). Foxl1 is a mesenchymal Modifier of Min in carcinogenesis of stomach and colon. Genes Dev 19, 311-315.

5. Stappenbeck, T. S., Mills, J. C. and Gordon, J. I. (2003). Molecular features of adult mouse small intestinal epithelial progenitors. Proc Natl Acad Sci USA 100, 1004-1009.

Research Advance #3

Elucidation of the mechanisms that underlie patterning of the undifferentiated embryonic gut tube to generate the individual digestive organs.

The GI tract and its evaginated derivatives (liver, pancreas, and biliary system) are a paradigm for inductive tissue interactions in development and, in particular, epithelial-mesenchymal interactions in organogenesis. Recent studies help define how undifferentiated endoderm is specified during embryogenesis in response to extraneous signals and the activities of tissue-restricted transcription factors, and indicate how these activities combine to confer tissue and organ properties. The identity of some tissue-restricted transcription factors that regulate gut development is known, though many others remain to be discovered. There is also growing (but still limited) understanding of chromatin states that distinguish the precursors of some embryonic digestive organs. In parallel, cancer biologists are gaining a better understanding of epithelial-stromal (mesenchymal) interactions in neoplasia, and the principles of developmental interactions are very likely to extend into the realm of tumor biology.

Selected citations:

1. Rossi, J. M., Dunn, N. R., Hogan, B. L. and Zaret, K. S. (2001). Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev 15, 1998-2009.

2. Bhowmick, N. A., Chytil, A., Plieth, D., Gorska, A. E., Dumont, N., Shappell, S., Washington, M. K., Neilson, E. G. and Moses, H. L. (2004). TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848-851.

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3. Kim, B. M., Buchner, G., Miletich, I., Sharpe, P. T. and Shivdasani, R. A. (2005). The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling. Dev Cell 8, 611-622.

4. Fujitani, Y., Fujitani, S., Boyer, D. F., Gannon, M., Kawaguchi, Y., Ray, M., Shiota, M., Stein, R. W., Magnuson, M. A. and Wright, C. V. (2006). Targeted deletion of a cis-regulatory region reveals differential gene dosage requirements for Pdx1 in foregut organ differentiation and pancreas formation. Genes Dev 20, 253-266.

5. Calmont, A., Wandzioch, E., Tremblay, K. D., Minowada, G., Kaestner, K. H., Martin, G. R. and Zaret, K. S. (2006). An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells. Dev Cell 11, 339-348.

Research Advance #4

Successful application of assorted model systems to investigate aspects of gut development that are best approached through biochemical, genetic, and developmental studies in diverse species, including Drosophila, chicken, and zebrafish.

Whereas the advances emphasized in Research Advances #1 through #3 are based largely on studies in the laboratory mouse, other model systems provide unique advantages and can lead to insights not readily obtained in mice. The zebrafish is a tractable model for combining “forward genetic” and chemical analysis of development; Drosophila embryos still hold some advantages for discovering pathway activities; and chick embryos are particularly amenable to manipulation of gene expression in vivo. Investigators have successfully exploited each of these properties to contribute to the growing understanding of mechanisms in GI tract development.

Selected citations:

1. Farber, S. A., Pack, M., Ho, S. Y., Johnson, I. D., Wagner, D. S., Dosch, R., Mullins, M. C., Hendrickson, H. S., Hendrickson, E. K. and Halpern, M. E. (2001). Genetic analysis of digestive physiology using fluorescent phospholipid reporters. Science 292, 1385-1388.

2. Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron, S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001). casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 15, 1493-1505.

3. Gaudet, J., Muttumu, S., Horner, M. and Mango, S. E. (2004). Whole-genome analysis of temporal gene expression during foregut development. PLoS Biol 2, e352.

4. Matsuda, Y., Wakamatsu, Y., Kohyama, J., Okano, H., Fukuda, K. and Yasugi, S. (2005). Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development. Development 132, 2783-2793.

5. Theodosiou, N. A. and Tabin, C. J. (2005). Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling. Dev Biol 279, 481-490.

6. Micchelli, C. A. and Perrimon, N. (2006). Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475-479.

2. GOALS FOR RESEARCH

Short-Term Goals (0-3 years): CAPITALIZE ON RECENT ADVANCES AND DEVELOP TOOLS TO INTEGRATE THE RAPIDLY EMERGING UNDERSTANDING OF GUT DEVELOPMENT.

1. Encourage research aimed to understand how particular cell/tissue niches are generated and maintained: regions of the endoderm that are fated to develop into pancreas, liver, biliary tree, and digestive tract; prospective fetal gut segments (esophagus, stomach and its sub-regions, duodenum, ileum, etc.); and the distinctive domains of small bowel crypts and villi.

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2. Develop tools that permit accurately targeted genetic studies in the mouse stomach and specific intestinal segments. It would be particularly useful to have a stable repertoire of transgenic mice that faithfully express the Cre recombinase, reporter genes such as GFP and -galactosidase, or toxigenes such as Diphtheria toxin, ideally in inducible forms.

3. Exploit the advanced understanding of the role of the Wnt-APC--catenin pathway in human colorectal cancer to develop new and effective treatment strategies.

4. Recruit (and retain) multi-disciplinary talent that is dedicated to answer important outstanding questions in gut development using diverse models and approaches.

Intermediate-Term Goals (4-6 years): DEVELOP A REASONABLY COMPLETE UNDERSTANDING OF THE PATHWAYS AND INTERACTIONS THAT MEDIATE CRITICAL PATTERNING EVENTS IN GUT ENDODERM AND GENERATE AND MAINTAIN THE SELF-RENEWING EPITHELIA OF THE STOMACH AND INTESTINE.

1. Develop a sophisticated appreciation of the major molecular pathways in development of the stomach, intestine, liver, and pancreas and attempt to relate these pathways to specific human diseases, including cancer. Scientific developments in colorectal cancer illustrate the value of such integration and suggest effective experimental strategies.

2. Improve understanding of how the major signaling pathways implicated in gut development interact with each other (the proverbial “cross-talk”) and determine how the particular confluence of ubiquitous signals helps generate specific structures and tissues.

3. Delineate the relative contributions of specific signaling pathways and transcription factors in gut development and study how the intersection between extraneous and cell-intrinsic signals mediates particular developmental processes.

4. Enable wide distribution of genetically engineered animal models that can be intercrossed or studied using different methods in different laboratories.

5. Integrate molecular databases (gene expression, chromatin-IP, cis-element analyses) with functional studies (siRNA, genetically engineered mice) to capture new pathways and to better appreciate the underlying circuitry.

Long-Term Goals (7-10 years): APPLY THE UNDERSTANDING FROM BASIC RESEARCH IN GI DEVELOPMENT TO DISSECT DISEASE MECHANISMS AND IDENTIFY TARGETS FOR THERAPY.

1. To identify the key factors required to specify each digestive organ and begin to appreciate how the knowledge may be applied to facilitate tissue regeneration in vivo or ex vivo.

2. To distinguish between factors whose functions are restricted to the developmental period and those that continue to influence critical activities in the adult organs.

3. Recognize the specific molecular defects associated with particular congenital malformations and with tissue metaplasias and cancer, especially Barrett’s esophagus, gastric intestinal metaplasia, intestine-type gastric cancer, pancreatic in situ neoplasia and adenocarcinoma, and a range of non-infectious hepatic disorders.

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3. MAJOR CHALLENGES AND STEPS TO ACHIEVE GOALS

Goal: Research aimed to understand how particular cell/tissue niches are generated and maintained.Challenge/Steps: (1) Develop powerful but faithful ex vivo approaches to replicate tissue niches in a manner that is amenable to experimental manipulation; (2) Detailed gene expression profiles of selected, highly enriched cell populations to identify molecular markers and candidate regulators.

Goal: To develop tools that permit accurately targeted genetic studies in the mouse stomach and specific intestinal segments.Challenge/Steps: (1) Validation of specific promoters/minigenes that drive tissue- or segment-specific gene expression; (2) Communication within the research community to identify, characterize, and distribute suitable mouse lines.

Goal: Capitalize on advanced understanding of the role of the Wnt-APC--catenin pathway in human colorectal cancer to develop new and effective treatment strategies.Challenges/Steps: (1) Small-molecule screens to disrupt protein-protein interactions; (2) Identification of additional pathway components such as kinases or other enzymes that may be more suitable candidates for targeting by small molecules.

Goals: (A) To develop sophisticated appreciation of the major molecular pathways in development of the stomach, intestine, liver, and pancreas; (B) To improve understanding of how the major signaling pathways implicated in gut development interact with each other; (C) To delineate the relative contributions of specific signaling pathways and transcription factors in gut development.Challenges/Steps: (1) Continued investment in sound investigator-initiated projects to delineate and study pathways in depth; (2) Detailed enumeration of the expression domains of specific signaling components at the level of RNA in situ hybridization on embryonic and adult tissue sections.

Goal: Functional evaluation of newly identified molecules (from gene expression, chromatin-IP analyses, etc.) in development of the GI tract.Challenges/Steps: (1) Develop innovative experimental approaches that permit functional evaluation in vivo and ex vivo.

Goals: To evaluate the extent to which molecules that mediate development of the GI tract are also active in adult gut and may be targets for therapy of specific disorders.Challenges/Steps: (1) Detailed enumeration of the expression domains of specific signaling components at the level of RNA in situ hybridization on embryonic and adult tissue sections; (2) Analysis of genetically engineered mouse strains at embryonic and adult stages, possibly in combination with selected Cre-expressing strains to enable tissue-specific recombination.

Goals: To recognize the specific molecular defects associated with particular congenital malformations and with tissue metaplasias and cancer.Challenges/Steps: (1) Working with tissue and tumor banks to study well-preserved and well-annotated clinical specimens for evidence of activation or disruption of specific pathways; (2) Generation of animal models of specified GI disorders to study molecular pathophysiology.

4. PATIENT PROFILE TOPIC

To be considered after the conference call.

5. GRAPHICS AND IMAGES


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NAME: Hannah Carey, PhD, University of Wisconsin School of Veterinary Medicine, MadisonWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Growth and Integrative Physiology

[Individuals contributing include: Denise Ney, Helen Raybould, Jeff Gordon lab (particularly Justin Sonnenburg and Peter Crawford)]

Research Advance #1Regulation of Intestinal Growth: Roles of Nutrients, Trophic Factors, and Neurohumoral Signaling

Recent evidence from animal models and human subjects with short bowel syndrome suggests that intestinal adaptive growth is regulated by several key hormonal mediators, including glucagon-like peptide-2 (GLP-2), insulin-like growth factor-I (IGF-I), epidermal growth factor and growth hormone. However, details of their mechanisms are still poorly understood, including the involvement of other cell types besides enterocytes in the intestinotropic effects. For example, recent reports support a role for neural regulation of GLP-2 action induced by enteral nutrients, with IGF-I acting as a downstream mediator. Better understanding of the cellular mechanisms responsible for intestinotropic hormone action may lead to improved treatments for individuals with intestinal failure who require parenteral nutrition.

Citations:

Estall, JL and Drucker, DJ. (2006). Glucagon-like peptide-2. Annu Rev Nutr 26:391-411.

Dube, PE, Forse, CL, Bahrami, J, and Brubaker PL. (2006). The essential role of insulin-like growth factor-I in the intestinal trophic effects of glucagon-like peptide-2 in mice. Gastroenterology 131:589-605.

Nelson, DW, Liu, X, Holst, JJ, Raybould, HE, and Ney, DM. (2006). Vagal afferents are essential for maximal resection-induced intestinal adaptive growth in orally fed rats. Am J Physiol Regul Integr Comp Physiol 291:R1256-R1264.

Martin, GR et al. (2006) Gut hormones, and short bowel syndrome: the enigmatic role of glucagon-like peptide-2 in the regulation of intestinal adaptation. World J. Gastroenterol. 12:4117-4129.

Short-Term Goals (1-3 yrs)

Obtain accurate measures of circulating and tissue concentrations of intestinotrophic in normal health and after bowel resection.

Localize expression of the intestinotrophic hormone in animal models and human subjects with short bowel syndrome before and after intestinal transplantation.

Intermediate-Term Goals (4-6 years)

Identify the intestinal stem cell populations that are upregulated by GLP-2.

Identify the nutrients that act synergistically with hormones to facilitate intestinal growth.

Characterize the neural pathways and downstream mediators that regulate GLP-2 action.

Evaluate the efficacy of intestinotrophic hormones given in conjunction with other GI drugs.

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Long-Term Goals (7-10 years)

Identify the major signaling pathways involved in intestinotrophic hormone action.

Demonstrate the efficacy of GLP-2 and downstream mediators of GLP-2 in clinical trials, including short bowel syndrome, inflammatory bowel disease, intestinal damage induced by cancer chemotherapy, and ischemic injury.

Steps To Achieve Goals

Develop an antibody that recognizes the bioactive intestinotrophic peptides. Understand cross-talk and synergism among intestinotrophic peptides, growth factors, nutrients, and

other growth-promoting molecules using animal models.

Research Advance #2Molecular Events Underlying Intestinal Growth and Adaptation

There has been significant progress in our understanding of the changes in gene expression and cell signaling pathways that are induced by the loss of intestinal mass, such as occurs during bowel resection and other gut injuries and during the adaptive response that follows. Maintenance of intestinal homeostasis during development and adult life is a complex process that requires a proper balance among cell proliferation, apoptosis, and differentiation, and involves interactions between epithelial and other cell types in the intestinal wall including mesenchyme and fibroblasts. Understanding the molecular pathways that mediate normal intestinal growth and the response to injury, and how extrinsic stimuli such as nutrients affect their activity is crucial for development of interventions to maintain intestinal mass and functional capacity. In particular, studies that have begun to elucidate the stem cell niche response following gut resection or injury (e.g., from ischemia, radiation, or trauma) may provide novel therapeutic targets to enhance gut mass and function.

Citations:

Sheng G, Bernabe KQ, Guo J, Warner BW. 2006. Epidermal growth factor receptor-mediated proliferation of enterocytes requires p21waf1/cip1 expression. Gastroenterology 131: 153-164.

Wang Y, Wang L, Iordanov H, Swietlicki EA, Zheng Q, Jiang S, Tang Y, Levin MS, Rubin DC. 2006. Epimorphin(-/-) mice have increased intestinal growth, decreased susceptibility to dextran sodium sulfate colitis, and impaired spermatogenesis. J Clin Invest 116: 1535-1546.

Brown SL, Riehl TE, Walker MR, Geske MJ, Doherty JM, Stenson WF, Stappenbeck TS. Myd88-dependent positioning of Ptgs2-expressing stromal cells maintains colonic epithelial proliferation during injury. J Clin Invest. 2007 Jan;117(1):258-69.

Helmrath MA, Fong JJ, Dekaney CM, Henning SJ Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice. Am J Physiol Gastrointest Liver Physiol. 2007 Jan;292(1):G215-22. Epub 2006 Aug 17.

Tang Y, Swietlicki EA, Jiang S, Buhman KK, Davidson NO, Burkly LC, Levin MS, Rubin DC. 2006. Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection. Am J Physiol Gastrointest Liver Physiol 290: G1280-G1288.

Erwin CR, Jarboe MD, Sartor MA, Medvedovic M, Stringer KF, Warner BW, Bates MD. 2006. Developmental characteristics of adapting mouse small intestine crypt cells. Gastroenterology 130: 1324-1332.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Henning+SJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Dekaney+CM%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Fong+JJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Helmrath+MA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Stappenbeck+TS%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Stenson+WF%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Doherty+JM%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Geske+MJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Walker+MR%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Riehl+TE%22%5BAuthor%5D


Determine downstream mediators of growth factor signaling (e.g., EGFR) that affect intestinal epithelial proliferation, and intrinsic as well as extrinsic death pathways (i.e., Bcl-2 family proteins).

Develop transgenic models that over expresses growth-promoting signaling molecules in the gut.

Develop cell-specific promoters to permit overexpression or deletion of critical gut stromal (myofibroblast) molecules.


Identify local and circulating factors that activate EGFR and other receptors associated with epithelial growth.

Determine the mechanism by alterations in stromal molecules affect DSS-induced colitis and other small bowel and colonic injury models.

Characterize the molecular basis of stromal-epithelial interactions in gut injury and repair to identify potential therapeutic targets, (using microarray and proteomics approaches for global gene and protein expression analyses)

Characterization of the cellular composition of the stem cell niche and alterations in niche-stem cell interactions in response to resection and regeneration.

Determine role of hedgehog signaling in normal intestinal growth and the response to resection


Target epimorphin and other mesenchymal-epithelial signaling pathways to enhance mucosal regeneration in inflammatory or ischemic conditions.

Develop therapeutic approaches that use the EGFR and other growth factor signaling pathways to enhance gut growth after resection.

Develop novel methods of tissue engineering utilizing knowledge of the stem cell and its niche, to create functional neomucosa.


Develop transgenic models with altered expression of growth-promoting molecules in health and disease states. Combine with appropriate models of abnormalities in intestinal morphogenesis/growth/ differentiation to assess relative roles of each.

Develop and maintain database of molecular mechanisms identified in intestinal growth and cell differentiation pathways. Include phenotypes of transgenic mouse models and results from other model organisms, including zebrafish, Xenopus, and Drosophila, that target specific genetic pathways and disease conditions.

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Research Advance #3Integration of Brain-Gut Signaling, Metabolism, and Mucosal Biology in the

Regulation of Body Mass

Considerable progress has been made in our understanding of the way in which the presence of nutrient stimuli in the gut lumen is sensed by endocrine cells and nerves. This information is crucial in the normal digestive processes that occur in the gut and may be altered in disease. The presence of luminal nutrients is also important in the short-term regulation of food intake. There is good evidence from both rodent models and from human studies that two GI hormones, CCK and PYY, are involved in the regulation of food intake via activation of neural substrates in the gut-brain axis. Moreover, long-term changes in the macronutrient content of the diet can alter the sensitivity of the gut-brain axis and thus may lead to long-term changes in body mass.

Citations:

Murphy, KG, Dillo WS, Bloom SR. 2006. Gut peptides in the regulation of food intake and energy homeostasis. Endocrinology Rev 27:719-727.

Stader, AD and SC Woods. 2005. Gastrointestinal hormones and food intake. Gastroenterology 128:175-191


Begin to develop technology to assess changes in the circulating gut hormone profile in fed and fasted states using animal models for weight gain/obesity. Develop approaches for identification/ quantitation of biomarkers using peptidomic, metabolomic, and other technologies.


Understand the interactions between adipokines and the gut-brain axis.

Determine the role of inflammatory mediators in the gut wall on the sensitivity of neural signaling in gut-brain axis.

Understand the mechanisms by which bariatric surgery leads to changes in body mass.


Develop effective, peripherally active substances for control of food intake and body weight using gut hormone-based therapies to target appetite circuits.

Develop therapeutic interventions to mimic the effects of bariatric surgery on body mass.


Use systems biology approach, integrating physiology with proteomics/metabolomics and other technologies to identify adipokines that influence gut function, and how signals originating from the gut affect adipose tissue biology and metabolism.

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Research Advance #4Role of the Nervous System in Gastrointestinal Inflammation

Interactions between the nervous and immune systems play important roles in normal and disease states in the GI tract. Recent research has provided new insights into neuro-immune relationships that may facilitate translation of basic science into therapeutic applications, particularly with regard to GI inflammatory diseases. One example is the cholinergic anti-inflammatory pathway, which modulates release of pro-inflammatory mediators in models of colitis, ischemia-reperfusion, postoperative ileus, and pancreatitis. Neuronal signaling pathways in the gut are also affected by inflammation. For example, pro-inflammatory cytokines can alter expression and function of the mucosal serotonin transporter (SERT), which affects neurohumoral signaling via serotonergic pathways. Changes in function of SERT and other neural pathways may therefore underlie the altered motility, secretion, and sensation seen in these inflammatory gut disorders. Better understanding of neuro-immune crosstalk in GI inflammatory disease is warranted; however, several fundamental questions must be addressed in order to advance this field.

Citations

Ghia JE et al. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology 2006 Oct;131(4):1122-30.

Tracey KJ. Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest 117: 289-296 (2007)

Altavilla D. et al. Activation of the cholinergic anti-inflammatory pathway reduces NF-κB activation, blunts TNF-α production, and protects againts splanchic artery occlusion shock. Shock. 2006 May;25(5):500-6.

Luyer MD et al. Nutritional stimulation of cholecystokinin receptors inhibits inflammation via the vagus nerve. J Exp Med. 2005,202(8):1023-9.

Foley KF et al. IFN-γ and TNF-α decrease serotonin transporter function and expression in Caco2 cells. Am J Physiol Gastrointest Liver Physiol 292: G779-G784, 2007.

von Boyen GBT et al. Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: A key to the regulation of epithelial apoptosis in Crohn's disease. Inflammatory Bowel Diseases 12 (5): 346-354, 2006.


Determine whether inflammation-induced changes in levels of neurohumoral mediators (e.g., protein, mRNA, tissue content, etc.) correlate with release of relevant mediators.

Identify the effects of manipulating afferent and efferent cholinergic pathways to the gut on cytokine release and downstream effects of cytokine action.


Identify the neural circuitry involved in pro- and anti-inflammatory pathways.

Understand the cause-and-effect relationships between inflammation and altered neural function. Does inflammation alter neural signaling, which subsequently leads to GI dysfunction, or are neuronal alterations associated with inflammatory states compensatory responses? If so, what role do they play in protection or restoration of gut function?

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Determine the functional implications of inflammation-induced changes in neural signaling—which changes are most relevant to function or disease progression?

Identify which aspects of the immune response in inflammatory states lead to changes in neuronal function and what intracellular signaling pathways are altered.

Understand the role of nutrition in animal models, including lipid-based diets, in the cholinergic anti-inflammatory pathway.

Begin development of therapeutics (drugs or devices) based on neuro-immune pathways targeted towards GI disease (e.g., IBD) and pathologies that have GI effects (e.g., shock, I/R injury).


Devise strategies to correct the molecular abnormalities in enteric neuronal function induced by inflammatory states, keeping in mind that some abnormalities persist for short, and others for longer, time periods after the initial insult.

Begin clinical trials with cholinergic agents, vagal stimulation, or pharmacologic manipulation of nicotinic receptors to activate the cholinergic anti-inflammatory pathway in patients’ disorders.


Develop multidisciplinary teams to address mechanisms responsible for neuro-immune protective and injurious states, including expertise in neuroanatomy/neurophysiology, immunology/inflammation, trauma, nutrition, and gastroenterology.

Use animal models of GI inflammatory conditions to manipulate neural signaling through pharmacological, electrical, or nutritional interventions; identify mechanisms of response and effects on morbidity/mortality.

Information obtained from non-neuronal models (e.g., epithelial cell lines) does not always translate to similar effects in neurons. Efforts must be made to determine whether mechanisms of neuro-immune communication in one cell type are the same, or differ, from other cell types. If not, studies must be directed to determine mechanisms appropriate for the relevant cells.

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NAME: Nicholas Davidson, MD, Washington University School of Medicine, St. LouisWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Digestion

OVERVIEW: Understanding the fundamental mechanisms and pathways by which nutrients, vitamins, and minerals are processed and assimilated by the digestive tract is key to developing approaches to disorders of the small intestine, biliary tract, and pancreas in which malnutrition is among the most prominent features and remains one of the least tractable. Advances in the molecular/genetic characterization of many of the dominant biochemical and physiological pathways by which macro- and micro-nutrients are digested and absorbed have resulted in a shift in our focus of considerations in regard to the pathophysiology of digestive disorders. By way of example, with the growing appreciation of the role of small intestinal transport functions, there has been a diminished emphasis on pathophysiological pathways that regulate luminal events in regulating digestive physiology. The last few years have witnessed increased understanding of many of the pathways involved by expanding a hierarchy of membrane transporters with distinct substrate specificity, as well as refined subcellular itineraries and destinations. Although the summary research advances outlined below have highlighted some spectacular new insights provided in regard to lipid and sterol transport, mention should be made of the continuing emergence of a role for peptide transporters, specifically hPEPT1 in the area of innate immunity, and transport of bacterial peptides (such as fMLP). In addition, there has been finer definition of intracellular acceptor molecules and nuclear hormone receptors that participate in metabolic channeling as well as nutrient sensing and that signal through both import and export pumps. Finally emerging new data strongly suggests that these conserved pathways for nutrient processing and delivery may serve heretofore unanticipated roles in innate immunity.


Research Advance #1Diversity in genetic pathways for absorption of cholesterol and other sterols

Identification of enterocyte membrane transporter (NPC1L1) specific for intestinal cholesterol uptake. Sensing and discrimination of subtle structural differences between cholesterol and plant sterols (sitosterol) and identification of sterol efflux pumps (ABCG5/G8) that minimize entry of non-authentic cholesterol through selective intestinal and biliary epithelial export into the lumen rather than into the systemic circulation. Identification of the basolateral cholesterol efflux pump ABCA1 and demonstration of its importance in the production of plasma HDL. Further advances have highlighted integrated regulation throughout the enterohepatic circulation in maintaining absorptive function. Collectively, these advances have greatly expanded our understanding of the complexities of whole body cholesterol homeostasis in health and disease.

Selected citations:

1. Altmann SW, Davis HRJr., Zhu L, Yao X, Hoos LM, et al. Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol absorption. Science 2004; 303: 1201 - 1204.

2. Davis HRJr, Zhu L, Hoos LM, Tetzloff G, Maguire M, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem 2004; 279: 33586 - 33592.

3. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, Hobbs HH. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 110:671-80, 2002.

4. Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B, Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest.;116:1052-62, 2006.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12208868&query_hl=5&itool=pubmed_DocSum

Research Advance #2Expanded understanding of developmentally regulated cell-cell communication pathways and

absorptive function

Understanding of the complexities of cell-cell signaling within the small intestine as a model for providing dialog in nutrient sensing, absorption and delivery into the systemic circulation. This includes newly recognized functions for integral structural proteins in establishing and preserving absorptive function. In addition, several lines of evidence point to unanticipated roles for Hedgehog signaling in intestinal nutrient (particularly lipid) absorption function. The mechanisms and pathways for these absorptive phenotypes are incompletely understood. A more complete understanding of how diverse developmental pathways modulate elements of absorptive function will have important implications for our understanding of integrated intestinal digestive physiology.

Selected citations:

1. Jones RG, Li X, Gray PD, Kuang J, Clayton F, Samowitz WS, Madison BB, Gumucio DL, Kuwada SK. Conditional deletion of beta1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality. J Cell Biol. 175:505-14, 2006.

2. Wang LC, Nassir F, Liu ZY, Ling L, Kuo F, Crowell T, Olson D, Davidson NO, Burkly LC. Disruption of hedgehog signaling reveals a novel role in intestinal morphogenesis and intestinal-specific lipid metabolism in mice. Gastroenterology. 122:469-82, 2002.

3. Wang CC, Biben C, Robb L, Nassir F, Barnett L, Davidson NO, Koentgen F, Tarlinton D, Harvey RP. Homeodomain factor Nkx2-3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera. Dev Biol. 15:152-67, 2000.

4 Madison BB, Braunstein K, Kuizon E, Portman K, Qiao XT, Gumucio DL. Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development. 132:279-89, 2005.

Research Advance #3Emerging hierarchy of ligands and receptors for intracellular signaling, metabolic

compartmentalization of nutrients

There has been new appreciation for the role of nuclear hormone receptors (FXR, LXR, PPARs) and other transporter/acceptor proteins (FABPs/FATPs) in energy sensing and in the maintenance of weight. In addition, new information concerning the metabolic compartmentalization of fatty acids (DGAT1/DGAT2) and monoglycerides (MGAT1/MGAT2) has provided new targets for obesity. Finally, advances in developing systems for understanding the dialog between host and luminal bacteria have expanded understanding of the relevance of the luminal bacterial environment in digestion and absorptive functions.

Selected citations:

1. Watanabe M, Houten SM, Mataki C, Christoffolete MA, Kim BW, Sato H, Messaddeq N, Harney JW, Ezaki O, Kodama T, Schoonjans K, Bianco AC, Auwerx J. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 439:484-9, 2006.

2. Houten SM, Watanabe M, Auwerx J. Endocrine functions of bile acids. EMBO J. 25:1419-25, 2006.3. Buhman KK, Smith SJ, Stone SJ, Repa JJ, Wong JS, Knapp FF Jr, Burri BJ, Hamilton RL, Abumrad

NA, Farese RV Jr. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis. J Biol Chem. 277:25474-9. 2002.

4. Yen CL, Farese RV Jr. MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine. J Biol Chem.;278:18532-7, 2003.

5. Stahl A, Gimeno RE, Tartaglia LA, Lodish HF. Fatty acid transport proteins: a current view of a growing family. Trends Endocrinol Metab.12:266-73, 2001.

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6. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA. 101:15718-23, 2004.

Research Advance #4Novel functions for genes involved in intestinal lipid absorption

A surprising series of discoveries has implicated conserved pathways in intestinal and hepatic lipid absorption in lipid antigen presentation and a role in innate immunity. In particular, emerging new information strongly suggests that the microsomal triglyceride transfer protein is responsible not only for lipidation of the export protein apoB, but also for lipid antigen presentation by CD1d. In addition, unanticipated findings have implicated apolipoproteins involved in lipid export (apoE) in lipid antigen presentation by CD1 molecules. It is worthy of inclusion in this category the possibility that the peptide transporter hPEPT1 may play a role in bacterial peptide presentation.

Selected citations:

1. van den Elzen P, Garg S, Leon L, Brigl M, Leadbetter EA, Gumperz JE, Dascher CC, Cheng TY, Sacks FM, Illarionov PA, Besra GS, Kent SC, Moody DB, Brenner MB. Apolipoprotein-mediated pathways of lipid antigen presentation. Nature.;437:906-10, 2005.

2. Dougan SK, Salas A, Rava P, Agyemang A, Kaser A, Morrison J, Khurana A, Kronenberg M, Johnson C, Exley M, Hussain MM, Blumberg RS. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells. J Exp Med. 15;202(4):529-39, 2005.

3. Brozovic S, Nagaishi T, Yoshida M, Betz S, Salas A, Chen D, Kaser A, Glickman J, Kuo T, Little A, Morrison J, Corazza N, Kim JY, Colgan SP, Young SG, Exley M, Blumberg RS. CD1d function is regulated by microsomal triglyceride transfer protein. Nat Med. 10:535-9, 2004.

4. Charrier, L, Driss, A, Yan, Y, Ndauti, V, Klapproth, JM, Sitaram, SV, Merlin, D. hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes. Lab Invest 86: 490-503, 2006.


Short-Term Goals (0-3 years): MAINTAIN AND EXPAND RESEARCH TOOLS AVAILABILITY AND INFRASTRUCTURE WITH ACCURATE AND HIGH-QUALITY PHENOTYPING

1. Encourage research programs aimed at generating and characterizing new and tractable models of digestive and absorptive functions in particular focusing on testing gain and loss of function phenotypes in defined genetic backgrounds.

2. Develop tools that will permit temporal and regional specific gain and loss of function testing of candidate genes in vivo, using inducible Cre-lox technology.

3. Identify cell membrane topology and biochemical mechanisms of known transporter function including substrate recognition, trafficking and regulation.

4. Identify mechanisms that control metabolic compartmentalization of substrates within the enterocyte and that regulate trafficking to distinct secretory pathways.

5. Encourage work that will identify and characterize novel genes that participate in the absorption of luminal substrates and xenobiotics.

Intermediate-Term Goals (4-6 years): DEVELOP A COMPREHENSIVE PROFILE OF INTESTINAL GENES THAT REGULATE MAMMALIAN ABSORPTIVE FUNCTIONS

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1. Extend studies of candidate genes to examining selected absorptive and metabolic pathways (eg cholesterol, bile acid, micronutrients) from human populations using humanized knock-ins of informative polymorphisms.

2. Develop targeted approaches to obesity, hyperlipidemia, diabetes, through testing candidate small molecule inhibitors of gene function using mouse and other models.

3. Integrate advances in developmental biology to understanding regional differentiation of intestinal absorptive function (eg ileal bile acid transporter, duodenal iron absorption) and possible plasticity.

4. Encourage development of selective siRNA and other tractable knockdown methodologies for widespread use in digestive/absorptive pathway interrogation.

5. Obtain more complete understanding of the dialog between host and luminal bacteria and the signaling pathways involved.

Long-Term Goals (7-10 years): TRANSITION FROM BASIC MOUSE AND OTHER ORGANISMAL MODELS INTO TESTABLE PATHWAYS WITH RELEVANCE FOR HUMAN DISEASE

1. Identify targeted therapeutics based on informative pathways that predict development of obesity, hyperlipidemia, diabetes.

2. Identify serum and tissue biomarkers that predict alterations in pathways identified above.

3. Recognize the specific molecular defects associated with nutrient malabsorption (including obesity) and defective or inappropriately increased intestinal nutrient delivery.

4. Initiate trials of selected pre and probiotics genetically selected to optimize luminal microbiome composition.


Challenge 1. Current animal models inadequate. Existing knockout models for intestine-specific deletion or transgene expression allow global intestinal lineage expression or deletion (villin) or mosaic patterns of expression (Fabp) while some promoter models allow duodenal expression at high levels (Adenosine deaminase). Needs include development of enteroendocrine specific promoters, ileal specific promoters. In addition, ability to direct siRNA knockdown to cells of the luminal GI tract would be major advance. Finally, mouse phenotyping is costly and resource intensive, precluding rapid development of useful functional databases.

Steps to achieve goal 1: Encourage development of needed animal and technical resources. Sharing of resources across programs, through center mechanisms as well as other centralized resources needs to be actively promoted and encouraged. Expand mouse phenotyping cores to digestive sciences. Encourage development of regional and national centers of excellence for methodologic development, for example with limited RFAs for pilot funding.

Challenge 2. Bioinformatics databases inadequate. Despite widespread use of arraying technologies, bioinformatics has lagged. This reflects complexities in developing algorithms for developing pathway based array outcomes and also the lack of trained human resource personnel. Development of new and useful mouse and other animal models could be linked to array-based phenotyping and proteomic surveying that would dramatically accelerate the pace of discovery.

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Steps to achieve goal 2: Invest in developing computational biologists and bioinformatics infrastructure. Encourage career development pathways for PhD scientists, not currently engaged in targeted areas of biomedical sciences, in order to generate useful approaches that can be rapidly disseminated into the scientific community.

Challenge 3. Population-based screening approaches and biomarker development is extremely high risk for individual investigators. Obesity, diabetes, and other disorders of nutrition and malabsorption are heterogeneous in origin and there is a paucity of well-characterized populations. Undertaking to develop long-range strategies for characterization of biomarkers currently beyond scope of academic investigators.

Steps to achieve goal 3. Encourage and support academia-industry dialog to support development of expanded understanding of human populations. Long-range efforts will require coordination through regional and national database development and appropriate serum and tissue banking. Could be opportunity for translational investigator development, if coupled with formal training, partnership with basic scientific program, loan forgiveness, etc.

4. PATIENT PROFILE TOPIC




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NAME: Marshall Montrose, PhD, University of CincinnatiWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Nutrient Fluid Absorption/Secretion

State of the Field

Using both functional and genetic approaches, many of the membrane transport proteins mediating intestinal salt, solute, and water transport were defined over the past 10 years. We are in the middle of defining the proteins that contribute to barrier function, in addition to revealing some surprising new pathways of intestinal fat and metal ion absorption. While there will be a continuing need to identify the proteins mediating and regulating absorption and secretion, the real challenge of the next decade is leveraging this information about the building blocks into an integrated view of epithelial function in health and disease. If the goal is maximal impact on human health, this cannot be done efficiently by separate investigations of tissue culture cells, animal modesl, and clinical outcomes. Experimental approaches must be integrated much the same as the experimental outcomes must be integrated.


Research Advance #1Blurring of Classical Boundaries of Absorptive and Secretory Cell Types

The intestinal epithelium is composed of a variety of cell types that are intermingled in the single layer of cells that separate the gut lumen from the body. Recent studies of intestinal epithelial development have revealed new pathways that regulate the balance among these different cell types (most notably Wnt signaling regulating epithelial proliferation, and Math-1 and Gfi in regulating the census of secretory cell types). With the constellation of absorptive and secretory proteins being defined, there is also increased ability to distinguish gradations of function. Recent studies have revealed surprising fluid and sodium absorptive functions in the structures of the colonic crypt, challenging our understanding of how the intestinal epithelium works in health and disease. Studies of epithelial repair and tissue remodeling in disease all emphasize the need to understand the complex interplay among epithelial cell types and their neighbors that results in the homeostasis of epithelial cell types (and thus epithelial function). It must be noted that the molecular pathway for intestinal epithelial differentiation to absorptive cell types has yet to be established. Our classic identification of intestinal epithelial cell types and functions is crude and limits our ability to understand deviations during disease and tissue remodeling.

Research Advance #2Regulation of Epithelial Barrier Function in Health and Disease

An accelerating body of work is aimed at understanding the molecular basis for barrier function thru elucidation of the function of claudins, occluding, and other tight junctional proteins. The regulation of tight junctions is now recognized as a major mechanism by which TNF causes diarrhea, providing a significant step forward in understanding a major symptom in IBD that brings patients to the clinic. The coordinate action of TNF on barrier function and absorptive transporters without effect on chloride secretion is a new concept and important for our understanding of inflammatory bowel disease. There is also expanding information about the specialized mechanisms by which intestinal epithelial cells actively contribute to body defense thru secretion of antibacterial peptides and repair of a disrupted epithelial layer. Complementary to understanding of barrier is the understanding of the molecular basis of cellular water transport. Cloning of water channel molecules in 1991 opened the door for advances in understanding cell and tissue fluid transport thru an expanding array of experiments that defined the impact of human aquaporin mutations on water channel function, crystal structure of aquaporins, and regulation of aquaporins. This led to the awarding of the 2003 Nobel prize in Chemistry to Peter Agre for discovery of the aquaporins. The advanced understanding of

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water channels will contribute greatly to understanding the pathways regulating barrier function versus fluid transport function.

Research Advance #3Gut Factors and Epi-Genetic Regulation of Food Intake and Energy Metabolism

Our understanding of cell and tissue regulation of intestinal transport has broadened in several notable areas. The discovery of non-mutational regulation of gene expression that can be passed through generations has ushered in an era of new understanding about disease predisposition. For example, studies have shown that the offspring of European women undergoing starvation during World War II are predisposed to a higher incidence of diabetes. These observations validate a need for more studies to evaluate this mode of regulation as a risk factor for obesity and other diseases that may involve changes in ion and nutrient transporters.

The study of nutrient absorption is now integrating information at the level of studying the regulation of food intake (satiety). Ghrelin is a recently discovered peptide hormone produced by the stomach that displays strong growth hormone-releasing activity and has a stimulatory effect on food intake and digestive function while reducing energy expenditure. Research in ghrelin has led to new insights into how this hormone produced by the stomach connects the endocrine control of nutritional homeostasis through the gut-brain interactions. Finally, epithelial cells have been shown to express Toll-like receptors (TLRs) for bacterial-epithelial recognition and signaling. This provides a hook to allow further studies how nutrients modulate epithelial/bacterial interactions in the gut.

Finally, recent research has revealed surprising regulation of intestinal iron absorption by the liver. It is widely recognized that iron homeostasis in the body is almost exclusively regulated at the level of intestinal iron absorption, because while the liver can store iron, the ability to excrete excess iron is poor. Prior to 2002, the main identified route to regulate iron uptake was iron regulatory elements on DNA and RNA that were sensitive to iron status, and affected the long-term response by modulating expression of proteins in the iron absorption pathway. Iron overload and iron deficiency syndromes have revealed unanticipated proteins originating from the liver that play a key role in the regulation of iron absorption. Hepcidin and haephestin were ascribed a role in iron absorption in 2002 and 1999, respectively, and ~500 publications have now explored these topics. Exploiting these discoveries will require a more integrated approach to understanding intestinal absorptive function than we can currently pursue, as well as a stronger understanding of the cell types mediating absorption and their response to specific stimuli.

Research Advance #4Intestinal Transporters Involved in the Absorption of Cholesterol and Fatty Acids

A surprising opportunity to explore an unexpected route for fat absorption came with the discovery of the drug Ezetimibe (generic name Zetia), which effectively inhibits cholesterol absorption. Since 2002, there have been ~400 publications on the subject. It is prescribed to patients to lower the amount of total cholesterol, LDL (bad) cholesterol, and apolipoprotein B (a protein needed to make cholesterol) in the blood. Zetia appears to bind to the Niemann-Pick C1-Like 1 protein (NPC1L1) on the gastrointestinal epithelium, a membrane transporter mediating cholesterol absorption. The decreased cholesterol absorption leads to an increase in LDL-cholesterol uptake into cells, thus decreasing levels of cholesterol in the blood plasma. If transporters are involved in the absorption of cholesterol and fatty acids (a new paradigm), then they are obvious targets for the development of drugs to combat the obesity epidemic. Both the cell types mediating cholesterol and fatty acid absorption by these new routes, and their response to specific stimuli, remain speculative.

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Short-Term Goals (0-3 years)The short-term goals all use existing research tools.

1. Define pathways mediating regulation of barrier function versus transport function.2. Identification of membrane transport proteins and intracellular chaperones of micronutrient and metal

ion absorption (iron, calcium, magnesium, etc.).3. The role of nutrients in modulating epithelial/bacterial interactions in the gut and bacterial ecosystems

within the gut lumen.4. Pilot test expanding use of non-mammalian models to studies of GI absorptive/secretory function

(e.g., zebrafish excellent for developmental studies, C. elegans good for candidate gene function analysis, drosophila mutant collection extensive).

Intermediate-Term Goals (4-6 years)The intermediate- term goals often require development of research tools and thus some enabling technologies must be initiated immediately to achieve these goals.

1. Appraise value and potential impact of outcomes from non-mammalian models prior to moving forward with more extensive programs.

2. Development of advanced mutant mouse models (tissue specific, knock-in, inducible mutations, humanized models) to minimize lethality of mutations, epi-effects driven by changes outside tissue of interest, adaptive responses, and to get animal models for studying human proteins.

3. Translate what the diversity of epithelial cell absorptive and secretory functions means at a proteomics level.

Long-Term Goals (7-10 years) The long-term goals all use the tools, concepts and information developed under intermediate goals.

1. Integrate information on role of cellular and protein diversity in creating efficient absorptive and secretory function in healthy tissue (human and mouse mandatory, non-mammalian systems as warranted).

2. Develop understanding of epithelial development and remodeling in response to injury, especially related to signals and pathways creating a balanced population of absorptive and secretory cells.

3. Understand the molecular and functional adaptation of individual epithelial cells of the intestine to challenge (surgery, inflammatory, diabetes, obesity, or experimental manipulation).


1. Lack of appropriate animal models . There are two issues here: (1) Most knockouts are constitutive and throughout the body; (2) Developing new mammalian models is slow. a) Need superior gene delivery methods for cells of intestinal tract and methods to study individual

transfected cells in situ.b) Develop mammalian models providing systems biology resolution. Such models would allow

integration of information from molecular regulation directly into studies defining organismal impact without needing to create new model (e.g., find role of protein without needing to create tissue-specific conditional knockout or transgene).i) Foster interdisciplinary research between gastrointestinal physiologists (including

immunologists and bacteriologists) and computational biologists.

2. Human workforce inadequate . Increasing emphasis on translational research needs cadre of trained, committed MD researchers and PhD researchers who can develop clinical research projects, as well as the committed involvement of bioinformatics researchers.

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a) Foster interdisciplinary research between gastrointestinal researchers and chemists and biomedical engineers.

b) Identify a mechanism to specifically encourage research that asks questions for both human and advanced mammalian animal models.i) Foster PhD graduate study in translational GI research.ii) Foster MD careers and training in basic research.iii) Assist investigators needing to reduce reliance on tissue culture models (via development of

novel animal models and/or re-training to use such models).

3. Increased success of translational research requires experimental approaches in animal models that can be more directly compared with human outcomes. Many experimental approaches to analyze cellular function not directly comparable between mouse and man. a) Develop equipment and chemical probes permitting parallel live tissue analyses in human

endoscopy and mouse intestinal tract.i) (REPEAT) Foster interdisciplinary research between gastrointestinal researchers and chemists

and biomedical engineersb) (REPEAT) Need superior gene delivery methods for cells of intestinal tract and methods to study

individual transfected cells in situ.

4. Genomic/proteomic approaches in GI research arenas are poorly developed. a) Establish centers for cell-type specific protein profiling, disease state profiling with standardized

procedures and outcomes. b) Develop a proteome fingerprint of cell types important to GI absorptive and secretory functions.

Must be performed in mouse and man, with some pilot testing in non-mammalian models.i) (REPEAT) Foster interdisciplinary research between gastrointestinal physiologists (including

immunologists and bacteriologists) and computational biologists.c) (REPEAT) Develop mammalian models providing systems biology resolution. Such models

would allow integration of information from molecular regulation directly into studies defining organismal impact without needing to create new model (e.g., find role of protein without needing to create tissue-specific conditional knockout or transgene).

4. PATIENT PROFILE TOPICS

Cystic fibrosis improvement in quality of life and life span informed by better understanding of CFTR function as a chloride channel and regulator of other membrane functions.

Obesity hardships improved by Zetia.

Oral rehydration solution improvements informed by basic science understanding of sodium-sugar cotransport function and stoichiometry.


Crystal structure of aquaporin water channel (request from Peter Agre, winner of 2003 Nobel Prize, along with a picture of Peter).

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NAME: Chung Owyang, MD, University of Michigan Medical Center, Ann ArborWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Neurophysiology and Endocrinology

Overview

In the last 5 years we have gained sophistication in our understanding of neuro-hormone control of gut functions and energy homeostasis. The unraveling of the complexity of signaling between diverse cells in the ENS provides the cellular and molecular basis for understanding some of the disorders affecting the enteric nervous system. The neurobiology of brain-gut interactions has become further characterized. This provides the necessary conceptual framework for scientist and clinician in their understanding and quest for new treatment of diseases and motility disorders. There is renewed and expanded interest in the role of the GI tract in the regulation of satiety and energy homeostasis. Increased understanding of the mechanisms governing nutrient sensing and peptide secretion by enteroendocrine cells allow investigators to exploit these pathways in their development of new agents to combat obesity and diabetes. A better understanding of the molecular mechanisms leading to disease and age-related apoptotic cell death provides hope for preventive and/or regenerative therapy. Finally a clear elucidation that neural crest stem cells persist in the adult gut and undergo changes in self renewal suggests that neuron replacement therapy can become a reality. Because of the diversity of the topic, it is difficult to cover all the major research advances in this area. The following are examples of the impact made as a result of basic and translational science research in the field.


Research Advance #1

The multiple constituents of the enteric nervous system (ENS) can have profound effects on its functioning. In addition to the nerves and smooth muscle cells, the normal functioning of the system requires participation of the interstitial cell of Cajal, glial cells, and enteroendocrine cells. For example, research on the interstitial cell of Cajal has dramatically altered the way we look at regulation of smooth muscle. Interstitial cells of Cajal pace gastrointestinal muscle by initiating slow waves in both muscle layers and appear to be the preferred sites for reception of neurotransmitters. These specialized cells in intramuscular layer also provide regenerative responses to and amplification of pacing messages from cells of Cajal in the myenteric plexus.

Recent studies indicate that interstitial cells of Cajal mediate mechanosensitive response in the stomach. Furthermore, lack of ICC in the pylorus explains distinct peristaltic motor patterns in the stomach and small intestine. These animal studies clearly indicate that it would not be possible to generate the motor program stored in the ENS without patterned electrical activity and synaptic connectivity provided by ICC. These findings have profound implications in human physiology and pathophysiology as abnormal networks have been reported in GI muscles from patients with motility disorders. These include achalasia, diabetic gastropathy, infantile pyloric stenosis, idiopathic gastric perforation, pseudo-obstruction and slow-transit constipation

Another example is the glial cells. In the CNS, glial cells have an important role in synaptic transmission plasticity and immunoprotection. It is likely that glia may play similar roles in the ENS. Recent studies show that ablation of the enterial glia in mice leads to a fulminent hemorrhagic jejunoileitis, suggesting a central role of the enteric glia in the maintenance of gut mucosal integrity. This possibility is further supported by the observation that development of enterocolitis occurs in mice after autoimmune targeting of glial cells. In human Crohn’s disease and experimental colitis in rats, the glial-derived neurotrophic factor (GDNF) is upregulated. This neurotrophic factor has strong anti-apoptotic effects on colonic epithelial cells which may be responsible for its protective action on the epithelial lining during mucosal inflammation.

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The smooth muscle cells not only are targets of inflammatory mediators they also secrete inflammatory mediators. For example, acid can activate vanilloid receptors (TRPV1) present on the squamous epithelial cells that release PAF. This may increase ROS generation in smooth muscle cells and in turn impair the LES tonic contraction. Catalase treatment restores the weak tonic contraction of circular muscle from a human donor with erosive esophagitis. Metabolic disorders may also cause motility disorders by directly affecting smooth muscle function. The high caveolar cholesterol levels in the smooth muscle cells in the gallbladder and stomach may impair muscle contraction, and restoring cholesterol to normal levels improves muscle contraction.

Citations:

1. Won KJ, Sanders KM, Ward SM. Interstitial cells of Cajal mediate mechanosensitive responses in the stomach. Proceedings of the National Academy of Science (USA) 102:14913-14918, 2005.

2. Wang XY, Lammers WJEP, Bercik P, Huizinga JD. Lack of pyloric interstitial cells of Cajal explains distinct peristaltic motor patterns in stomach and small intestine. Am J Physiol 289:G539-G549, 2005.

3. Steinkamp M, Geerling I, Seufferlein T, von Boyen G, Egger B, Grossmann J, Ludwig L, Adler G, Reinshagen M. Glial-derived neutrophic factor regulates apoptosis in colon epithelial cells. Gastroenterology 124:1748-1757, 2003.

4. Clerc N, Gola M, Vogalis F, Furness JB. Controlling the excitability of IPANs: a possible route to therapeutics. Curr Opin Pharm 2:657-664, 2002.

5. Gianino S, Grider JR, Cresswell J, Enomoto H, Heuckeroth RD. GDNF availability determines enteric neuron number by controlling precursor proliferation. Development 130:2187-2198.

6. Rossi J, Herzig KH, Voikar V, Hiltunen PH, Segerstrale M, Airaksinen MS. Alimentary tract innervation deficits and dysfunction in mice lacking GDNF family receptor alpha2. J Clin Invest 112:707-716, 2003.

Research Advance #2

During the last decade accumulating laboratory and clinical evidence indicate that IBS is a real clinical entity and not a psychosomatic disorder. Much progress can be attributed to (i) a better characterization of the neurobiology of brain gut interactions. The corticotropin-releasing factor (CRF) signaling pathway perhaps is the best described brain gut circuit closely related to the pathogenesis of IBS (ii) advances in neuroimaging to identify the brain regions responsible for perception and modulation of visceral afferent signals from the upper and lower GI tract ,and (iii) elucidation of the roles of peripheral serotonin/5HT receptor signaling system in the ENS.

The CRF signaling pathway coordinates endocrine, behavioral, and immune responses to stress. Activation of CRF receptor 1 and 2 produces differential effect on gastric and colonic motility. Clinical studies show that stimulation of CRF1 pathways produces the key symptoms of IBS diarrhea-predominant patients. These symptoms are alleviated by CRF1 receptor antagonists supporting the involvement of the CRF1 system at the central and peripheral sites in the pathogenesis of IBS triggered by stress. The CRF1 receptor antagonist that is directed at normalizing a sensitized CRF system holds great promise for a variety of stress-related GI disorders including IBS and cyclical vomiting syndrome.

Another major advance is the ability to image the living human brain with various neuroimaging modalities. This has greatly enhanced our ability to study brain gut interactions in health and in disease conditions. The brain regions involved in conscious perception of sensory information coming from the peripheral (insular cortex) have been identified and should be contrasted with the dorsal anterior cingulated cortex (dACC) which mediates the effective response and motivational drive. The magnitude and gain of signals going to these regions is highly influenced by central arousal and cortico-limbic systems. Symptom components of IBS can be dissected and attributed to specific areas of the brain that mediate cognitive, emotional and motivational component of the discomfort. This new approach to the study of functional GI disorders provides more insightful

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information on the pathophysiology of this group of disorders. The ability to study a neurobiological substrate with imaging modalities rather than relying on highly variable subjective symptoms (e.g., Rome criteria) makes it possible to investigate the role of genetic factors and receptor physiology on the pathophysiology of symptoms. We should have a precise endpoint to evaluate therapeutic interventions on distinct brain networks involved in afferent processing and modulation. Meaningful results from such studies can be obtained from much smaller samples of subjects compared with epidemiological or traditional pharmacological studies.

Lastly the characterization of the peripheral serotonin/5HT receptor signaling system in the ENS represents another significant advance in our endeavor to treat functional bowel disorders. Multiple 5HT receptor subtypes (5HT1, 5HT2, 5HT3 and 5HT4) are present in the ENS. The differential distributions of 5HT receptor subtypes make it possible to use 5HT3 antagonists and 5HT4 agonist to treat motility disorders and IBS. When all the signaling by 5HT is over, its action is terminated by uptake into enterocytes or neurons that is mediated by the serotonin reuptake transporter (SERT). A number of investigators have found that the colonic mucosa of IBS patients had reduced expression of SERT (mRNA and immunohistochemistry), whereas the number of enteroendocrine cells and the release of serotonin under baseline conditions or in response to stimulation are normal. Thus the serotonergic network appears to play a key role in the neurohormonal brain-gut axis in health and disease, and should be an important target for new therapeutic approaches.

Citations:

1. Tache Y and Bonaz B. Corticotropin-releasing factor receptors and stress-related alterations of gut motor function. J Clin Invest 117:33-40, 2007.

2. Martinez V, Tache Y. CRF1 receptors as a therapeutic target for irritable bowel syndrome. Curr Pharm Des 12:4071-4088, 2006.

3. Gravanis A and Margioris AN. The corticotrophin-releasing factor family of neuropeptide in inflammation: potential therapeutic applications. Curr Med Chem 12:1503-1512, 2005.

4. Mayer EA, Naliboff BD, Craig ADB. Neuroimaging of the brain-gut axis: from basic understanding to treatment of functional GI disorders. Gastroenterology 131:1925-1942, 2006.

5. Morgan V, Pickens D, Gautam S, Kessler R, Mertz H. Amitriptyline reduces rectal pain related activation of the anterior cingulated cortex in patients with irritable bowel syndrome. Gut 54:601-607, 2005.

6. Hobson AR, Furlong PL, Worthen SF, Hillebrand A, Barnes GR, Sign KD, Aziz Q. Real time imaging of human cortical activity evoked by painful esophageal stimulation. Gastroenterology 128:610-619, 2005.

7. Berman SM, Chang L, Suyenbu B, Derbyshire SW, Stains J, Fitzgerald L, Mandelkern M, Hamm L, Vogt B, Nalboff BD, Mayer EA. Condition-specific deactivation of brain regions by 5HT3 receptor antagonist alosetron. Gastroenterology 123:969-977, 2002.

8. Hicks GA, Coldwell JR, Schindler M, et al. Excitation of rat colonic afferent fibers by 5HT3 receptors. J Physiol 544:861-869, 2002.

9. Gershon MD. Plasticity in serotonin control mechanisms in the gut. Curr Opin Pharmacol 3:600-607, 2003.

10. Coates MD, Mahoney CR, Linden DR, et al. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology 126:1657-1664, 2004.

Research Advance #3

Until recently, it was generally believed that neural crest stem cells (NCSCs) undergo progressive restrictions in developmental potential and terminally differentiate soon after reaching post migratory sites. The postnatal peripheral nervous system was thought to lack stem cells. A number of recent studies show that NCSCs persist in the adult gut and they undergo changes in self renewal. Functionally the postnatal gut NCSCs make neurons that express a variety of neurotransmitters but

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lost the ability to make certain subtypes of neurons that are generated during fetal development. These include serotonergic and adrenergic neurons. This may be due to the loss of responsiveness of postnatal gut NCSCs to the neurogenic effects of BMPs. It should also be noted that cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. For example upon transplantation of uncultured NCSCs into developing peripheral nerve in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus cell fate in the nerve is stem cell determined. Although we are still a long way from using stem cell technology to do replacement therapy, the demonstration that neural crest stem cells persist in the adult enteric nervous system and undergo self renewal opens up a new possibility for regeneration after injury or disease.

Citations:

1. Kruger GM, Mosher JT, Bixby S, Joseph N, Iwashita T and Morrison S. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential and factor responsiveness. Neuron 35:657-669, 2002.

2. Bixby S, Kruger GM, Mosher JT, Joseph NM, Morrison SJ. Cell intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35:643-656, 2002.

3. Joseph NM, Mukouyama YS, Mosher JT, Jaegle M, Crone SA, Dormand EL, Lee KF, Meijer D, Anderson DJ and Morrison SJ. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells. Development 131:5599-5612, 2004.

Research Advance #4

The recognition of the pivotal role of gut hormone in glucose homeostasis has opened up new therapeutic options in the treatment of type 2 diabetes mellitus. Among the various gut hormones, both GLP1 and GIP promote insulin biosynthesis and islet cell survival. Additionally, GLP1 also inhibits glucagons secretion and gastric emptying and induces satiety. GIP engages receptors on adipocytes coupled to energy storage. On the other hand, CCK and gastrin do not seem to acutely regulate levels of plasma glucose but might be important for stimulating the formation of new cells by stimulating islet neogenesis. These pleiotropic actions of gut hormones may be exploited to develop novel therapeutics in the treatment of disorders of energy homeostasis.

Recognition of the GI tract’s crucial role in satiety signaling and control of energy homeostasis, body weight, glucose homeostasis and other metabolic systems is important in the formation of new approaches to combat obesity. Most of the satiation and orexigenic peptides are found in the GI tract. These include ghrelin, GLP1, PYY, oxyntomodulin and urocortin. Rationale manipulation of the neuroendocrine pathways regulating appetite may be used to treat obesity. For example, bariatric surgery works primarily by physiological rather by mechanical mechanisms. There is a close relationship between gastric emptying and regulation of hunger, satiety and body weight. Gastric bypass is associated with markedly suppressed ghrelin levels possibly contributing to the weight reducing effect of the procedure. The recent demonstration that bile acids induce energy expenditure by promoting intracellular thyroid hormone activation may have great physiological and therapeutic significance. Postprandial concentrations of bile acid should be sufficient to stimulate cAMP production and one of the genes encoding type 2 deiodinase enzyme which converts T4 to T3. In this manner bile acids could be hormonal signals linking food intake to diet induced increases in metabolic rate. This provides a potential approach to fighting obesity. Furthermore gut microbiota can be an important contributing factor to the pathophysiology of obesity. Metagenomic and biochemical analyses show that mouse gut microbiota have different capacities to harvest energy from the diet. This trait is transmissible as colonization of germ-free mice with an “obsese microbiota” results in a significantly greater increase in total body fat than colonization with a “lean microbiota.” This

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observation if confirmed in humans will strongly suggest that gut microbiome may be a biomarker, a mediator, and a new therapeutic target for people suffering from obesity.

Citations:

1. Drucker DJ. The role of gut hormones in glucose homeostasis. J Clin Invest 117:24-32, 2007.2. Ahren B et al. Inhibition of dipeptidyl peptidase-4 reduces glycemia sustains insulin levels, and

reduces glucagons levels in type 2 diabetes. J Clin Endocrinol Metab 89:2078-2084, 2004.3. Zander M, Madsbad S, Maden JL and Holst JJ. Effect of 6 week course of glucagon-like peptide 1 on

glycemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel group study. Lancet 359:824-830, 2002.

4. Meier JJ et al. Stimulation of insulin secretion by intravenous bolus injection and continuous infusion of gastric inhibitory polypeptide in patients with type 2 diabetes and healthy control subjects. Diabetes 53 (Suppl 3): S220-S224, 2004.

5. Rooman I, Bouwens L. Combined gastrin and epidermal growth factor treatment induces islet regeneration and restore normoglucemia in C57 B16/J mice treated with alloxan. Diabetologia.

6. Farilla L et al. Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology 144:5149-5158, 2003.

7. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 443:289-295, 2006.

8. Batterham RL. Inhibition of food intake in obese subjects by peptide 443-36. N Engl J Med 349:941-948, 2003.

9. Turnbaugh PJ, Ley RE, Mahowald MA, Margrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027-1031, 2006.

10. Miyawaki K, Yamada Y, Ban N, et al. Inhibition of GIP signaling prevents obesity. Nature Medicine 8:738-42, 2002.

11. Cummings DE, Weigle DS, Frayo RS, et al. Plasma ghrelin after diet-induced weight loss or gastric bypass surgery. N Engl J Med 346:1623-30, 2002.

12. Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 439:484-89, 2006.


Short-Term Goals (1-3 years)

The structural and functional organization of ENS continues to evolve. We need to more fully characterize the enteric neural networks; understand the cross-talk between the enteric neurons, glial cells, interstitial cell of Cajal, smooth muscle cells, and enteroendocrine cells; and define the signal processing within the ENS in response to nutrient, mechanical stimulation, inflammation, metabolic stress, and changes in bacterial flora. Technically, we must develop the tools to visualize state of activity of relevant cells (ICC, enteric neurons, muscle cells, and neuroendocrine cells) in a live context of tissue (in vitro or in vivo), organ and system (in vivo) and develop quantitative analysis of spatio-temporal patterns.

We must investigate the molecular and electrophysiological characteristics of the various cellular components of the ENS. This includes identification of receptors, channels and signal transduction systems unique to different cell types in the ENS as they may be targets for new drug developments to treat motility disorders.

Among the neurons in the ENS, there is much to learn about the interneurons. Available evidence suggests they mediate motility reflexes and are the real site of integration. We need to identify the molecular and electrophysiological characteristics of these neurons and learn how they process information and coordinate sensory and motor reflexes. To visualize the state of activity of these

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neurons in live tissues and organs may help us to understand their roles in initiating specific motility patterns.

In the CNS, glial-derived neurotrophins and neurotrophic factors such as GDNF have strong effects on neurite outgrowth and differentiation and are able to protect neurons from apoptosis under various inflammatory conditions. It is likely that glial cells may play similar roles in the ENS. This needs to be thoroughly investigated. The mechanisms responsible for upregulation of the GDNF in inflammatory bowel conditions should be examined and its cellular actions to promote epithelial restitution should be investigated. Alterations of this novel gut neuroepithelial crosstalk may contribute to more severe course of disease in IBD.

Because the intrinsic primary afferent neurons (IPANs) are the initiating neurons for enteric reflexes, they are strategically placed to influence the intensity of the reflexes. We need to fully characterize the plasticity of the channels, receptors, and intracellular control systems in response to inflammation, metabolic stress, and changes in luminal bacterial flora. Altered properties of IPANs might be involved in the development of intestinal hypersensitivity and changed motility in IBS. This should be evaluated using an appropriate animal model and subsequently in human tissues.

Currently little is known about the site and mechanisms by which alterations in blood glucose levels is sensed. The glucose sensing neurons in the CNS are unlikely to play a major role in mediating postprandial digestive and metabolic function in response to physiological changes in circulating glucose as the glucose level in the CSF only ranges between 10-30% of blood glucose level. Comprehensive studies must be performed to investigate the peripheral mechanisms of glucose sensing. The site is likely to be at the vagal afferent fibers that may mediate gastric motility, satiety, and hepatic glucose uptake and outputs. Abnormality of this pathway may occur in patients with diabetes and/or obesity.

The GI tract clearly plays a crucial role in satiety signaling and control of energy homeostasis. It is important to define the molecular basis for chemo- and mechano-reception in the gut to sense the ingested nutrient environment. At the same time we also need to have a better understanding about the interaction between nutrients and microbes sensing mechanisms in the gut.

Characterize alterations in the gut-based 5HT signaling system in IBS and motility disorders. This may include SERT, modulators of SERT expression, and number of enterochromaffin cells and contents. The mechanism responsible for diminished serotonergic signaling in inflammation should be investigated. Transcripts encoding tryptophan hydroxylase-1 and SERT can be decreased. Successive potentiation of 5HT and/or desensitization of its receptor could account for the symptoms seen in diarrhea-predominant and constipation-predominant IBS, respectively. This possibility should be investigated.

Progress in the research of functional bowel disease is partly hampered by lack of suitable animal models to mimic the disease conditions. Although animal models of visceral hypersensitivity are available, they are not ideal as the pathophysiology may be quite different. We need to establish scientific criteria to determine the validity of animal models for IBS and other motility disorders.


Define the roles of various cellular components of the ENS in the mediation of physiological events such as motility, sensory transmission, secretion, and blood flow. There is a need to integrate cellular events with whole system physiology.

Our understanding of ICC pathology is still in its infancy, but a picture is beginning to emerge that many disparate motility disorders result in loss of ICC. One approach is to make large-scale screenings of genetic changes that occur in tissues undergoing loss of ICC and investigate whether there are

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common genetic fingerprints that either produce or result from loss of ICC. Pathophysiological models coupled with genomics analyses may offer the opportunity to discover why ICC is vulnerable in apparently disparate motor disorders and how to recover ICC networks in disease organs.

Identify the distinct neural circuits involved in mediating different motility patterns. Define the principle receptors, neurotransmitters, and synaptic connections for each circuit. Identify the “switch” responsible to change one motility pattern to another. Examine how mechanical stimulation, metabolic stress, and inflammation can alter these circuits. This type of information may be important to define the pathophysiology of IBS.

Over the last decades, numerous molecules, pathways, and mechanisms linking the brain and the gut are now emerging but there is as yet comparatively little understanding of how these may be involved in organ dysfunction and disease. From neurochemical substrata and pathways that have been dissected, we have yet to find out which are utilized in health and whether these differ in disease and, if so, whether such differences are causes or merely consequences of the disorder. Expanded research effort should aim at unraveling the pathophysiology of brain-gut interactions.

We need to have a better understanding of the molecular mechanisms underlying age-related apoptosis of ENS neurons. Contemporary techniques for probing genetic and proteomic changes that occur with age, such as the use of oligonucleotide microarray and protein chip technologies, will help the field to evolve from phenomenology to hypothesis-driven research. It is important to establish the mechanisms that maintain the integrity of the adult ENS and its capacity to respond to altered function or “plasticity” in adulthood and old age. Studies of neurotrophic and growth factors and the associated signaling pathways may provide some clues for this type of investigation.

We should continue our efforts to identify distinct brain circuits involved in autonomic regulation, appetite control, pain perception, and emotional and cognitive modulation. Characterize the networks in healthy control populations and various patient populations.

Further studies are needed to understand the cellular and molecular mechanisms of neuro and endocrine bidirectional communication between the GI tract and CNS to regulate body weight and metabolic function. At the same time a better understanding of the physiological and molecular mechanisms by which bariatric surgery improves body weight and metabolic regulation are needed. This might shed light on novel mechanisms we can manipulate to treat obesity.

Obesity is a result of the interaction of environmental and genetic factors that mediate energy intake and expenditure. The molecular mechanism linking adiposicity to overnutrition remains unknown. Recent literature suggests GIP is a key molecule linking overnurtition to obesity. Excessive fat intake induces hypersecretion of GIP, which increases nutrient update and triglyceride accumulation at the adipose sites. This can readily cause obesity and hyperinsulinemia. Hyperinsulinemia will further increase nutrient uptake into adipose sites, completing a vicious cycle of developing adiposicity. Further studies should be performed to explore the possibility of GIP abnormalities in patients with overnutition and obesity.

Investigate the channels and signal transduction pathways that mediate cell mass, resistance to apoptosis, and stimulation of biosynthesis and secretion of insulin. Agents with these biological properties may become therapeutics to treat diabetes.

Over the last decade, there were many major advances in the understanding of the molecules that regulate the development of the ENS. However, much remains to be learned about how the various signals interact to yield a “normal enteric nervous system.” We should continue to search for clues as we begin to identify developmental defects resulting in neuronal loss such as in Hirschsrpung’s disease and hypertrophic pyloric stenosis. We need to identify molecules and pathways that promote

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proliferation and differentiation of enteric neurons and/or guide the growth of enteric axons to their targets.


Genomic analysis of animal model of ICC loss may demonstrate common endpoints for the fate of ICC in a variety of motility disorders. From these studies, it may be possible to determine specific gene profiles that occur in tissues that have suffered ICC loss or in the tissues in the process of losing ICC. This information may allow scientists to design specialized tests that reliably evaluate the status of ICC networks from biopsy material. Development of a molecular test that could pick up pathological changes more reliably and at earlier time points may be highly beneficial as we may be able to halt the degeneration and minimize the damage and loss of function.

The fate of ICC in variety of motility disorders is unknown. Cells may undergo apoptosis or redifferentiation into muscle cells. Understanding the molecular mechanisms responsible for differentiation and redifferentiation of ICC may allow scientists to perform tissue engineering and to restore functional populations of ICC in patients with ICC loss.

Following identification of distinct brain circuits responsible for various GI functions and pain perception, we should characterize the signaling systems and receptors within these neural circuits using PET ligand imaging in rodents and in humans. Ultimately, we should be able to correlate the individual circuits identified with symptom production in patients (“intermediate phenotypes”) and establish correlation with distinct genotypes that include genome-wide search for polymorphism and haplotypes.

With the goals established above, ultimately we need to be able to fully characterize the pathomechanisms underlying symptom generation in common functional and motility disorders, including identification of genetic and early environmental influences and natural course of the disease. This should lead to more effective therapeutic intervention.

Degeneration in the enteric nervous system resulting in motility disorders is one of the hallmarks of aging and can be a major determinant of quality of life for the aged. Neuron replacement therapy should be our targeted goal. Once we have obtained the blueprint of the key neural circuits in the ENS, we should utilize our knowledge gained in developmental biology and organ genesis to manipulate the neural crest stem cells in the bowel wall to proliferate and differentiate and provide the signaling molecules to guide the growth of enteric axons to their targets.

It is clear from recent data that the bile acid-cAMP-D2-T3 pathway has an important physiological role in energy expenditure and homeostasis. Administration of bile acid to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of bile acids is critically dependent on the induction of Type II iodothyronine deiodinase (D2). This factor is critical to convert the minimally active precursor of thyroid hormone T4 into the potent T3 form. Additional studies should be performed to investigate the possibility of polymorphism in the D2 gene which may result in altered whole body glucose disposal rate, resulting in obesity.

Obesity research remains the top priority for investigators in the United States. The capacity to adjust food intake in response to changing energy requirements is essential for survival and good health. We need to continue to characterize the molecular, cellular, and behavioral mechanisms that link changes of body fat store to adaptive adjustments of feeding behaviors. We need to define the diverse blood-borne and affective neural signals that transmit information regarding nutrient status and energy stores to the brain where it is integrated with cognitive, visual, olfactory, and taste cues. Understanding the complexity of this energy homeostasis system will then allow us to start to search for mutation of key molecules mediating these pathways in patients with severe obesity.

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General Comments

Advances in the molecular biology sciences and proteomics represent an unprecedented opportunity for us to understand key biologic pathways pertaining to the neuroendocrine system of the GI tract. However, much remains to be done. The limitations of NIH funding, hesitation of young trainees to embark on a scientific career, the lack of a cohesive national research agenda, and the inability to translate observations made at the bench to bedside practice all contribute to the challenges before us.

The search for novel technologies and development of national core facilities should be encouraged. We should put major emphasis on the development of new technology to visualize states of activity or relevant cells in live context of tissues, organ, and system and develop quantitative analysis of spatio-temporal patents. Application of bioinformatics to motility disorders and further refinements of proteomics and metabolic technologies to the gut and motility will be important. The development of multi-institutional consortium agreements to share resources should be fostered. This will help us take advantage of national core resources laboratories for expansive technologies such as neuro-imaging, genotyping of microflora, and advanced cell and in-vivo imaging techniques. Emphasis on translational research approaches cannot be overstated. The funding agencies should insist that the studies mechanism have a clear relevance for clinical medicine.

To best utilize our limited talent and financial resources, in the most effective manner, we need to develop a national agenda of priorities and fund research programs that have different clinical themes. A consortium of research teams consisting of scientists with different backgrounds and expertise from different disciplines working together to solve a common problem should be developed. These researchers may be recruited from institutions and laboratories with different skills to contribute to solving a clinical disorder. The research should be complementary, interdisciplinary, and coordinated with a common purpose and targets. This approach may be far superior to the current use of funding silos of purely specialty-based research. This will foster thinking outside the box and promote creative solutions for many perplexing issues in neurogastroenterology.

Goal: To integrate cellular events with whole system physiology and pathological conditions. Challenge/Steps: The “bench to bedside” approach is difficult to achieve by any single laboratory. This requires interdisciplinary research effort. Research on the pathophysiology of GI motility disorders continues to be hampered by lack of tissue from patients who are carefully genotyped and phenotyped. Generation of large national and international data and tissue banks may help to address this issue.

Goal: To understand how pathways linking brain and gut may explain organ dysfunction and disease. Challenge/Steps: Neuroplasticity and redundancy of pathways may obscure the physiological and/or pathophysiological significance of the brain-gut axis. Need animal models and watch for compensatory responses over time.

Goal: Redundancy of both GI and CNS pathways governing energy homeostasis poses major challenges for scientists designing anti-obesity drugs.Challenge/Steps: Treatment of obesity may require drug combinations that target discrete components of energy homeostasis, satiety, or food reward system.

Goal: To understand the physiology of functional bowel disease and search for new therapeutic optionsChallenge/Steps: IBS is a heterogeneous group of disorders with different pathophysiology for different subgroups. Currently there are no biomarkers and therapeutic target. Patients need to be better phenotyped based on clinical symptoms and pathophysiology consideration. Need to

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understand cellular mechanisms of disease to identify drug targets. The same is true for most motility disorders.

4. PATIENT PROFILE TOPICPlease give brief description of an idea for a patient profile relevant to this research topic.

None

5. GRAPHICS AND IMAGES If you have access to the graphic/image, please send as email attachment; if not, please provide information on the source of the graphic/image.

None

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NAME: Abigail Salyers, PhD, University of Illinois at Urbana-ChampaignWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Intestinal Microbiota and Digestive Health

(Contributors: J. I. Gordon, L. Hooper, S. Giovannoni, G. Olsen, S. Finegold)

1. RESEARH ADVANCES

Research Advance #1Genomic analysis of the microbial population of the human colon

The microbial population that normally inhabits the human colon is one of the densest microbial populations known (ca. 1012 per gm), accounting for at least 30% of the volume of colon contents. It is also highly complex, containing hundreds of bacterial species. The numerically predominant species are all obligate anaerobes, such as Bacteroides spp. and a number of poorly characterized gram positive species. This complex population is acquired shortly after birth and persists in the colon throughout life.

The colonic microbiota clearly has a major impact on human health. This impact includes such proven effects as its role in human nutrition, in the stimulation of mucosal cell turnover, suppression of intestinal pathogens such as Clostridium difficile, and as a reservoir for antibiotic resistance genes. Members of the microbiota that are normally innocuous are also significant causes of post-surgical infections and infections in cancer patients. Less clearly established are a number of suspected but still unproven links to such conditions as inflammatory bowel disease, colon cancer, and obesity.

In the past, testing hypotheses about the effects of the colonic microflora on human health has been hampered by the fact that researchers had to rely on cultivation-based methods for characterizing the colonic microbiota, an approach that was both time-consuming and unreliable. The increased speed and economy of genomic analysis has now made it possible to answer a number of important questions, particularly those concerning the role of the microbiota in digestive diseases such as inflammatory bowel disease and colon cancer.

Two main molecular approaches are currently being applied to characterize the intestinal microbiota. They are sequencing of bacterial 16S rRNA genes, which provides information about the species composition of the microbiota, and metagenomics, which uses shotgun sequencing to obtain information about the physiological potential of the microbiota. Taking a census of the species in the microbiota by sequencing of 16S rRNA genes is an important first step in characterizing the microbiota and is the approach that is most likely to provide near-term advances in understanding the microbiota. However, prior experience with this approach in soil and marine biology has underscored the importance of standardizing and validating this approach so as to facilitate accurate interpretation of results.

Citations:

Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. 2005. Host-bacterial mutualism in the human intestine. Science 307:1915-1920.

Eckburg, PG, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. 2005. Science 308:1635-8.

Hayashi H, Takahasi R, Nishi R, Sakamoo M, Benno Y. 2005. Molecular analysis of jejunal, ileal, cecal and rectosigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J. Med. Microbiol. 54:1093-101.

Kibe R, Sakamoto M, et al. 2005. Movement and fixation of intestinal microbiota after administration of human feces to germfree mice. Appl. Environ. Microbiol. 71:3171-8.

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Short Term Goals (1-3 years)

1. Collect and organize a central, carefully curated database to accommodate the mass of 16S rDNA sequence data that will be generated.

This undertaking will include, as a first step, a thorough reevaluation of the sequences used for amplification of the rDNA molecules. Most of the primer sets currently in use were developed over a decade ago, when the databases were much smaller and were heavily biased toward pathogenic bacterial species and thus did not reflect the true diversity found in bacterial populations. Second, this sequence collection should be curated by eliminating primer sequences, chimeric sequences and poor quality sequences, all of which distort the result obtained when sequences are analyzed.

2. Develop a microarray that contains rDNA sequences from all of the major human colonic species. Develop reliable methods for isolating rRNA from fecal material and hybridizing it without amplification to the microarray.

Bypassing the amplification step would remove the amplification bias that can distort representation of different species. The availability of good quality microarrays of this type would make it possible to analyze more easily and cheaply the number of samples that will be needed to establish connections between population composition and digestive diseases. An important caveat is that the microarrays need to be made inexpensive enough to allow many different laboratories to have access to them.

3. Determine the extent of person-to-person variation and variation with age and diet in healthy people.

This information will not only reveal how much variation the human colon has evolved to tolerate but will be important in designing comparative studies of people with disease and healthy people.

4. Refine and validate the “humanized” mouse model in which germ-free mice are colonized with human fecal material.

Such a model will be particularly useful in the future, now that many mouse models for intestinal disease are becoming available, and will remove the variables introduced by the fact that laboratory mice normally have different microbiota than humans.


1. Establish the effect of location in the small intestine and colon on the composition of the microbiota.

Initial studies can be done in mice, but human samples, though difficult to obtain, will be essential to validate the results of animal studies. The small intestine should be an important part of this work, with special emphasis on the adherent micirobiota. It is well established that mice have an adherent small intestinal microbiota that consists mainly of a filamentous bacterium that is probably a Clostridium species, but the existence of such a population in humans is controversial. Given the fast flow of contents through the small intestine, it would be surprising if an adherent microbiota did not develop as the predominant microbiota. There may also be an adherent microbiota in the colon.

2. Explore new technologies that would allow relatively non-invasive collection of specimens from the human intestinal tract.

For example, one can imagine a capsule that would open when triggered by a transmitter to trap bacteria on a matrix that would “freeze” the bacteria by killing them chemically in a way that

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preserves their DNA and RNA. It might even be possible to develop a capsule that would detect surface properties of bacteria directly and transmit signals to a receiver outside the body.

3. Launch pilot studies of the association of microbiota shifts with such digestive diseases as inflammatory bowel disease and colon cancer. Use these results to decide at what level (species, genus) to decide that a significant difference had been discovered.

Long Term Goals (7-10 years)

1. Perform large studies on the connection between the species composition of the colonic microbiota and such conditions as inflammatory bowel disease, colon cancer. Such studies may require multiple samples from the same person.

Information about the species composition of the microbiota of people with a disease condition or a pre-disease condition (e.g., colonic polyps associated with later development of colon cancer) will at best establish an association. To prove cause-and-effect, it will be necessary to show that controlled changes in the microbiota prevent a disease or improve the prognosis. Antibiotic interventions would be one way to accomplish this goal, but feeding bacteria (the probiotic approach) might also be able to change the microbiota sufficiently to determine whether a clinical improvement or effective prevention has occurred. This latter approach could help solve a long-standing clinical problem, the repopulstion of the bowel microbiota of patients who have taken antibiotics or have undergone cancer chemotherapy. Note that this type of intervention will involve confronting a number of ethical issues, but such interventions have been done in the case of the vaginal microbiota (to prevent preterm birth) and the prevention of diseases such as heart disease by antibiotic therapy.

Anticipated Translational Outcomes

Rational design of mixtures of probiotic bacteria that could be used to repopulate the intestines of people who had been treated with antibiotics or chemotherapeutic agents and were experiencing adverse side effects. Such mixtures might also be able to restore the normal microbiota in people experiencing microbiota shift diseases, if indeed such diseases as inflammatory bowel disease and colon cancer are caused by microbiota shifts.

Development of rapid diagnostic methods to assess the composition of the major groups of colonic bacteria, which might be used to detect pre-disease conditions with a simple noninvasive method such as a rectal swab so that preventive interventions could be initiated.

Development of diagnostics that would make it feasible to track effects of therapies that might affect the composition of the microbiota. For example, such diagnostic technology could be used as part of the evaluation of potential side effects of different modes of administration of new antibiotics such as disruption of the microbiota or increases in resistance to antibiotics.

Research Advance #2:Moving from species composition to microbiota function

Taking a “census” of the bacteria that are present in the colon at any particular time is important, but this approach has an important limitation. The species identity of a microbe does not usually reveal its metabolic potential because of the incredible metabolic diversity of bacteria. The metabolic potential of a microbial population can be assessed in two ways. First, genome sequences of cultivated members of the microbiota can be obtained and analyzed. This approach is limited by the fact that it will miss members of the microbiota that have not been cultivated. Even in the case of cultivated members, the cultivated strains may not be representative of the majority of strains of that species. Second, a metagenomic analysis can be done, in which DNA isolated directly from a bacterial population is determined by

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random sequencing. This approach currently is limited by the difficulty of assembling random sequences into whole genomes, but advances in bioinformatic technology should reduce this problem.

The information obtainable from either of these approaches is limited by the fact that there are still many genes of unknown function. Further advances in bacterial physiology and recognition of new motifs will help to reduce this number. Microarray analysis of gene expression will also help.

An interesting possible side outcome of metagenomic studies may be to ask the question: is there a normal viral microbiota of the colon? It is clear that many colonic bacteria carry bacteriophages, some of which carry genes encoding toxins that cause human disease. If eukaryotic viruses are routinely being shed by human intestinal cells, their genes might also be detected as part of a metagenomic analysis, especially if a soluble fraction of colon contents were used as the source of DNA. Of course, detection of viruses would depend on how abundant these viruses are in the ecosystem and also on our ability to recognize viral sequences.

Citations:

Gill SR, PopM, Deboy RT et al. Metagenomic analysis of the human distal gut microbiome. 2006. Science 312:1355-9.

Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027-1031.

Venter JC, Remington K et al. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66-74. (Note: Although this article focuses on analysis of a marine ecosystem, it shows what can be done by way of sequence assembly into partial genomes and also shows some of the problems that can be encountered.)


1. Obtain multiple genome sequences of the “unknown majority” of colonic bacteria, the gram positive anaerobic bacteria.

Although many of these species, which comprise at least 60% of all colonic bacteria, have been cultivated, cultivation is difficult. Obtaining the genome sequence of the leading representatives of this group of bacteria, whose numbers are undisputedly large but whose roles in the colon are unknown, would help to develop hypotheses about their metabolic potential.

2. Begin to collect and organize the newly emerging mass of metagenomic data.

This goal is a much greater computational challenge than organizing rDNA sequences. More needs to be known about such important areas as culling out genes from contaminants and assembling genomes.

3. Undertake a systematic and carefully controlled metagenomic analysis of the NORMAL human intestinal (and perhaps murine intestinal) population. Results of rRNA gene census results will be important for guiding the design of such a study.

4. Develop methods for systematic screening of large numbers of bacterial enzyme activities in colon contents (metabolome).

This area has been neglected of late but is an important supplement to the microarray analysis of mRNA levels because these levels do not always give an accurate reflection of enzyme activities, which are the most important endpoint in terms of effects on the human body. Batteries of enzyme

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assays have been developed for some bacterial species (e.g., Biolog). Initially, it is probably not feasible to develop truly comprehensive screens of bacterial activities, but the results of metagenomic analyses should suggest a more limited battery of enzyme activities that could serve as the first use of this type of analysis. Guidance from the high throughput strategies being developed by the biotechnology industry to detect enzyme activities could serve as a source of technology.

5. Develop bioinformatic analytical procedures to assess the significance of the differences between metagenomes obtained in connection with different dietary, disease, and age conditions.


1. Continue to expand the metagenomic analysis, now extending it to various diets and disease states.

2. Begin to develop microarrays based on the emerging metagenomic data and the individual genome sequences, and – most importantly – make these arrays available to a wide variety of scientists including nutritionists as well as clinicians.

3. As a supplement to the systematic screening of bacterial enzyme activities, develop screening procedures for bacterial toxins and hormone-like compounds. A bacterial toxin called E. coli ST toxin has been shown to have hormone-like action on intestinal cells but no further searches for similarly acting bacterial peptides has been done.


1. Early metagenomic, microarray, and enzymatic analyses will undoubtedly demonstrate associations between changes in diet or various disease conditions and changes in various genes and activities of the microbiota. Develop approaches for testing a cause-and-effect relationship by examining interventions, such as antibiotic treatment or repopulation of the bowel of people whose microbiota has been affected by antibiotic or other treatments.


Rational design of prebiotics, chemicals intended to restore normality to the activities of the intestinal microbiota or even improve the protective effects of the microbiota.

New diagnostic techniques would make possible a new type of nutritional intervention that takes into account not only such things as micronutrients and caloric values but also the effects of different diets on the function of the microbiota.

New diagnostic techniques may also help to identify pre-disease states at a time when intervention is still effective.

Research Advance #3Evaluating the composition and functions of colonic end product users, the archaea and sulfate-

reducing bacteria

The main focus in discussions of the human colonic microbiota has been on the numerically predominant populations and clinically significant minor populations such as the enterics and enterococci. An often overlooked minor population consists of fermentation end-product users such as the methanogenic archaea and the sulfate-reducing bacteria. These microbes use hydrogen and carbon dioxide from the fermentation of dietary polysaccharides and sulfate from fermentation of such host-produced polysaccharides (e.g., mucins and mucopolysaccharides) as carbon and energy sources.

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These end-product utilizers have two possible roles in the functioning of the colonic microbiota. First, their consumption of hydrogen, carbon dioxide, and sulfate increases the efficiency of the colonic fermentation of polysaccharide. Second, their products such as methane and sulfide have potential effects on the human body. Methane is readily absorbed across the colonic mucosa and in about one-fifth of the population is excreted in breath at levels high enough to be easily detectable. Sulfide is a poison at high concentrations but could also have more subtle effects on colonic cells at low concentrations.

Citations:

Attene-Ramos MS, Wagner ED, Plewa MJ and Gaskins HR. 2006. Evidence that hydrogen sulfide is a genotoxic agent. Mol. Cancer Res. 4:9-14. (Note: This paper focuses on the mutagenic effects of sulfide from sulfate reducers but also summarizes some evidence for toxic effects of sulfide from colonic bacteria on the colonic mucosa.)

Samuel BS, Gordon JI. 2006. A humanized gnotobiotic mouse model of host-archael-bacterial mutualism. Proc. Natl. Acad. Sci. 103:10011-6


1. Develop new primers for amplifying the 16S rRNA genes of methanogenic archaea and sulfate reducing bacteria. Take a census of the normal human colonic microbiota using these primers. Metagenomic studies may not yield sequences from these bacteria because of their relatively low numbers, but genes from these microbes other than 16S rRNA, such as genes involved in methanogenesis or sulfate reduction, could be amplified so that their diversity could be assessed.


1. As an adjunct to studies of possible associations between the predominant bacterial populations and various disease states, include primers for the rRNA or other genes in the studies.

2. Develop microarrays and enzyme assays to assess the activities of these microbes. In the case of microarrays, selective amplification of the mRNAs from genes of interest would probably be needed, but enzyme activities might be detectable directly due to the high activity of these microbes in some people. Assess possible associations between gene expression/enzyme activities and digestive diseases.

3. Assess the effects of methane and sulfide on intestinal cells using the humanized mouse model.


1. These goals are dependent on what is found in the earlier years. If interesting associations are found, explore possible therapies or other interventions such as diet on disease status.


By focusing only on the major intestinal populations, we may be looking in the wrong place for members of the microbiota that cause or exacerbate digestive diseases. If these end-product utilizers are involved, different approaches to interventions will have to be used.

If the colonic fermentation contributes to obesity, these minor populations, which may well have an impact on the efficiency of absorption of products of the colonic fermentation and thus the contribution of the microbiota to digestion.

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Research Advance #4Assessing the extent and significance of horizontal gene transfer among human colonic bacteria.

Bacteria in the colon interact genetically as well as metabolically. Bacteria, unlike humans and other mammals, do not experience species limitation and can and do transfer DNA across species, genus, and phylum lines. This has been demonstrated repeatedly in the laboratory. Only recently has evidence begun to emerge that such broad host range transfers actually occur with appreciable frequency in natural settings. The colon seems to be a hotbed of this type of transfer activity. This has so far been demonstrated in the case of transfer of antibiotic resistance genes and transfer of virulence factors, but may involve other types of genes as well. Horizontal gene transfers may go beyond bacteria that normally reside in the colon. They could also affect bacteria that are merely passing through the colon such as swallowed oral bacteria or soil bacteria, which can then be excreted into the environment and transmitted to other hosts and other sites.This is the reason colonic bacteria have been called reservoirs of resistance and virulence genes.

The broadest host range transfers of these genes appear to be mediated by conjugative elements that are often quite large (> 50 kbp in size) and can be integrated into the chromosome (conjugative transposons) or replicate autonomously as plasmids. Bacteriophages also transfer genes unrelated to their replication cycle, such as genes that encode protein toxins that can cause human disease. These transfers have been assumed to occur mostly within species or between closely related species, but questions about this limitation have been raised recently. Not only do conjugative elements and phages transfer DNA encoding such functions as antibiotic resistance and toxin genes but environmental conditions that stimulate increases in copy number (e.g., during the bacteriophage lytic cycle) can also increase expression of genes like toxin genes. This is thought to occur in the case of E. coli O157:H7 in which the phage-carrying toxin genes are induced to enter lytic phase by some types of antibiotica, thus producing more toxin. Evidence is also beginning to emerge that regulatory genes on transmissible elements may affect the expression of genes outside the element itself.

Some impacts of these activities of transmissible elements are obvious. For example transfer of antibiotic-resistance genes increases the incidence of resistant bacteria and makes it more likely that opportunistic infections caused by intestinal bacteria will be very difficult to treat. Transfer of virulence genes like toxin genes spread the ability of bacteria to cause disease. There may well be other impacts, however, that have yet to be discovered. An interesting possibility is suggested by a bacterial toxin, E. coli ST toxin that has hormone-like properties and actually uses the receptor for a human hormone, guanylin. The large size of many transmissible elements means that they can transfer a very large number of genes in one transfer event.

The conditions that stimulate transfer mobile elements are only beginning to be understood. Understanding what conditions stimulate transfer of mobile elements may help to prevent the rise in the number of strains that are resistant to antibiotics or produce virulence factors that aid them to cause disease.

Citations:

Franco AA. 2004. The Bacteroides fragilis pathogenicity island is contained on a putative conjugative transposon. J. Bacteriol. 186:6077-92.

Hasegawa F, Shimonishi Y. 2005. Recognition and signal transduction mechanism of Escherichia coli heat stable enterotoxin and its receptor guanylate cyclase C. J. Pept. Res. 65:261-271.

Salyers AA, Gupta A, Wang Y. 2004. Human intestinal bacteria as reservoirs of antibiotic resistance genes. Trends Microbiol. 12:412-6.

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Shoemaker NB, Vlamakis H, Hayes K, Salyers AA. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl. Environ. Microbiol. 67:561-8.

Waldor MK, Friedman DI. 2005. Phage regulatory circuits and toxin gene expression. Curr. Opin Microbiology 8:459-65.


1. Mobile elements have only been examined in a limited number of colonic bacteria, mostly gram negative bacteria such as the Bacteroides spp. and the enterics. Mobile elements in the gram-positive anaerobes that comprise the majority of the colonic microflora should be examined in depth. These mobile elements may be revealed by examining genome sequences from these bacteria.


1. Seek to understand all of the activities encoded by known mobile elements found in colonic bacteria. This is not as daunting a task as it might seem because there is a considerable amount of sequence conservation, at least among some of the genes in question. The goal is to be able to identify potential mobile elements in the genome sequences that will be emerging from the genome sequences of single bacteria and the metagenomic data.

2. Use microarray technology to determine whether and how genes carried on mobile elements are regulated, especially by commonly encountered environmental stimuli such as antibiotics, disinfectants and pollutants. There are examples of regulation of gene expression by antibiotics and indirect evidence for effects on gene expression by disinfectants and metals.

3. Seek to identify factors that stimulate or suppress transfer of mobile elements so that interventions can be designed to prevent transfer as well as gene expression.


1. Determine what effects products of mobile elements, such as toxins and hormone-like compounds, have on human intestinal cells. Knowing this would allow the design of antitoxic drugs that might be used as treatments of these disorders.

2. Determine whether intestinal bacteria can transfer DNA to intestinal cells, especially crypt cells, which are not terminally differentiated. Bacteria have been shown to be able to transfer DNA to eukaryotic cells such as yeasts in the laboratory. Whether transfer to mammalian cells occurs is not known. Integration of foreign DNA, such as mammalian virus DNA, is known to cause cancer. If bacterial DNA can enter and integrate into intestinal cell genomes, such events might lead to cancer.

Anticipated Translational Outputs

The ability to design antibiotic administration regimens that reduce the spread of antibiotic-resistance genes could help limit the rise of resistant bacteria that might later act as opportunistic pathogens.

If bacteria produce toxins or hormone-like compounds that contribute to digestive diseases, compounds could be developed that block the binding of these bacterial products to intestinal cells.

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NAME: Warren Strober, MD, National Institute of Allergy and Infectious Diseases, NIHWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Mucosal Immunology

(Contributors: M. Boirivant, E. Butcher, J. Holmgren, N. Lycke, L. Hooper, T. MacDonald, A. Macpherson, M. Neurath, and D. Podolsky)

Overview:

The mucosal immune system encompasses the constellation of immune mechanisms that enable the function of the immune system at the site of its greatest exposure to the microbial environment. Some unique features of this system arise from its juxtaposition to an enormous commensal microbial community that constitutes a quasi-independent biome existing within the very confines of its mammalian host. While this microbial flora helps to protect the organism from invasion by mucosal pathogens and hones the responses of the mucosal immune system to such pathogens, it creates the possibility of mucosal immune over-reaction and the most important immune diseases of the mucosa, the inflammatory bowel diseases of the gastrointestinal tract. Other unique features of the mucosal immune system arise from the related fact that this system is intimately associated with a vital epithelial cell layer that at once forms a physical barrier to the entry of organisms (both commensal and pathologic) and serves as an active participant in the mucosal response.

These unique features define the goals of research in mucosal immunology for the next ten years. By the end of the coming decade we can reasonably hope to have mastered the techniques of enhancing mucosal immune responses so as to prevent or ameliorate acute and chronic infections of the mucosal surface, including the vexing infection caused by the human immunodeficiency virus, an infection that is now rightly recognized primarily as a mucosal infection. In addition, we can anticipate a major advance in the understanding of the factors that cause inflammatory bowel disease and the development of definitive approaches to the treatment of these diseases.

Research Advance #1The role of the intestinal microflora in the maintenance of mucosal immune homeostasis

As mentioned in the overview, the mucosal immune system is unique in that it lies in close proximity to an enormous consortium of commensal organisms that play multiple roles in maintaining gut homeostasis, including the prevention of colonization by pathogens and in the promotion of epithelial cell repair following damage. These organisms are separated from mucosal lymphoid elements by a single layer of epithelium and an over-lying mucus that prevents wholesale entry of the bacteria. Nevertheless, it has shown that commensal organisms do enter the mucosa via Peyer’s patches and are picked up by dendritic cells in these lymphoid structures. An additional mode of entry of commensal organisms is via CD11c+ dendritic cells in the lamina propria that extent processes between epithelial cells and take up organisms; this process is enhanced in epithelium exposed to TLR ligands. The function of such limited commensal uptake is several fold. First it leads to the production of IgA antibodies that functions to limit further uptake of organisms by coating organisms and preventing colonization. Second, it leads to the induction of regulatory T cells that control T cell responses to commensal antigens in the mucosal lumen and thus prevents the mucosal flora from inducing inflammation.

Citations:

Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., and Medzhitov R (2004) Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229-241.

Macpherson, A.J. and Uhr, T. (2004) Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303:1662-1665.

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Chieppa, M., Rescigno,M. Huang, A.Y. and Germain, R.N. (2006) Dynamic Imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J.Exp. Med., 203:254-258.

Tringe, S.G., von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, M., Podar, J.M., Short, E.J., Mathur, Detter, J.C., Bork, P., Hugenholtz, P and Rubin, E.M. (2005) Comparative metagenomics of microbial communities. Science 308:554-557.


Further advances in the understanding of the relationship between the commensal organisms will depend in part on a better definition of the nature of the organisms in the mucosal flora in health and disease. Recently, there has been a revolution in the technical ability to characterize the gut flora with high output molecular techniques. Use this methodology to define possible difference in the mucosal flora among healthy individuals and individuals with inflammation or neoplasms of the GI tract.

Intermediate Term Goals (4-7 years)

Simultaneously track the growth of multiple organisms following introduction into an axenic environment and thus to define the effects of individual members of the microflora on mucosal immune responses.

Use molecule techniques of defining commensal bacteria to define the progression of bacterial colonization occurring in the newborn gut in healthy babies and those with gastrointestinal diseases.

Extend the above study of commensal microflora to murine models of gastrointestinal inflammation to better characterize the organisms that contribute to the prevention/induction of inflammation.

Gain a better understanding of the differences between innate responses to commensal organisms and pathogens so as to elucidate why commensal organisms generally lead to protective and anti-inflammatory responses in normal individuals and pathogens lead to inflammation.

Long Term Goals (7-10 years)

Identify the antigens and TLR ligands that are specific for particular organisms and how they affect gut mucosal immune function at the level of innate and adaptive immune responses.

Develop approaches to the manipulation of commensal microflora populations so the population protects the host from the development of infection and inflammation and or reverses on-going inflammation.

Research Advance #2The role of epithelial cells in mucosal host defense and inflammation

It is now apparent that epithelial cells are not passive participants in the mucosal immune response, but on the contrary, play active and perhaps key roles in the shaping/initiation of that response. This manifests itself in the ability of epithelial cells to produce chemokines and cytokines that initiate innate immune cell responses and thus set up a first line of defense against the intrusion of organisms into the mucosa. Many of these responses are induced by Toll-like receptors and nucleotide oligomerization domain-LRR receptors interacting with microbial components. There is also increasing evidence that epithelial cells produce substances such as thymic stromal lymphopoietin (TSLP) that influence dendritic cell function and thus determine the nature of T cell differentiation that occurs in relation to mucosal antigenic stimulation. On another level, epithelial cells transport IgA via the polyimmunoglobulin receptor and IgG via the neonatal Fc receptor and in doing so carry anti-bacterial agents to the luminal surface and/or move

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immunoglobulin/antigen complexes in a bi-directional manner across the epithelium. Finally, epithelial cells produce a variety of anti-bacterial substances including defensins (cryptins) and lectins that regulate the bacterial population in intestinal crypts and thus contribute to the development of inflammatory bowel disease. Regarding the latter, there is initial genetic evidence that susceptibility genes in IBD act through effects on the barrier function of the epithelium so that changes in the way the epithelium controls the composition and density of the local commensal flora and/or allow the penetration of flora into the lamina propria, may influence the development of inflammation in the underlying mucosa.

Citations:

Benn, M.C. Darfeuille-Michaud, A., Egan, L.J., Miyamoto, Y. and Kagnoff, M.F. (2002) Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-kappa B and MAP kinase pathways and the upregulated expression of interleukin 8. Cell Microbiol. 4:635-648.

Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A., Sampietro, G.M., Nespoli, A., Viale., G., Allavena, P., and Rescigno, M (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature Immunol. 6:507-514.

Yoshida, M., Kobayashi, K., Kuo, T.T., Bry, L., Glickman, J.N., Claypool, S.M., Kaser, A., Nagaishi, T., Higgins, D.E., Mizoguchi, E., Watatsuki, Y., Roopenian, D.C., Mizoguchi, A., Lencer, W.I. and Blumberg, R.S. (2006) Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria. J. Clin. Invest. 116:2142-2151.

Cash, H.L., Whitham., C.V., Behrendt C.L., and Hooper, L.V. (2006) Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313: 1126-1130.

Cario E., Gerken, G., and Podolsky, DK (2004) Toll-like Receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127:224-238.


Elucidation of the intra-cellular signaling pathways involved in the production of chemokines, cytokines, and cryptins produced by epithelial cells in response to commensal and pathogenic organisms.

Definition of the factors that regulate the expression of TLR and NLR on/in epithelial cells and the effect of stimulation of these receptors on epithelial barrier function, chemokine/cyokine production, and cryptin production.

Exploration of the role of the neonatal Fc receptor in mucosal immune responses and induction of mucosal unresponsiveness.

Elucidation of the factors produced by epithelial cells including TSLP, IL-10, and TGF-beta that effect dendritic cell function and/or T cell differentiation.


Production mice expressing epithelial cell-specific knock-outs of key genes involved in epithelial mucosal immune function and barrier function followed by extensive evaluation of the effect of the loss of these genes on gut homeostasis, particularly with respect to understanding IBD and/or infection of the GI tract.

Definition of the function of genes affecting epithelial cell immune function or barrier function identified as susceptibility genes in IBD.

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Characterization of embryonic and adult stem cell differentiation into epithelial cells focusing particularly on the acquisition of properties that relate to epithelial immune functions. Characterization of the differentiation of specialized epithelial cells such as M cells and Paneth cells.

Development of methods for the long-term cultivation of primary epithelial cells and the generation of mice with humanized epithelial cells.

Development of robust and reproducible model systems for the elucidation of epithelial cell-intestinal lymphoid cell interactions.

Research Advance #3The role of antigen-presenting cells in the mucosal immune system

The dendritic cell is a key cellular player in the mucosal immune response; as such it plays a role in mucosal host defense and in the pathogenesis of inflammatory bowel disease. Studies of the function of the mucosal dendritic cell revealed that these cells are, as a population, somewhat unique. For example, in mice the CD11chi population is made up of several sub-populations, including a CD8lo sub-population that have an increased propensity to produce IL-10 and stimulate Th2 responses as compared to phenotypically similar cells in the spleen; in addition, a CD11clo (plasmacytoid) dendritic cell population is present in the gut, but in this environment does not produce IFN-; finally, there are the aforementioned dendritic cells that sample antigens in the mucosal lumen via the extension of dendrites. Dendritic cells in the Peyer’s patches process antigen released from infected and apoptotic epithelial cells and then present these antigens to CD4+ T cells; dendritic cells in the lamina propria, perhaps those that engage in luminal antigen sampling, migrate to draining lymph nodes for presentation of antigens to T cells which then demonstrate a unique propensity to migrate back to the mucosa; these dendritic cells (as well as those from the Peyer’s patches can also migrate back to the lamina propria where they take part in the differentiation of B cells into IgA producing cells through the elaboration of B cell differentiation factors such as Baff and APRIL. Finally, evidence has recently emerged that mucosal dendritic cells may be uniquely involved in the induction of regulatory T cells in the mucosa via the production of TGF- as well as the induction of Th17 producing cells via production of IL-6 and TGF-. They thus control the balance of effector cells and regulatory cells at mucosal sites.

Citations:

Iwasaki, A., Kelsall, B.L. (2000) Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine. J.Exp.Med; 191:1381-1394.

Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A., Sampietro, G.M., Nespoli, A., Viale., G., Allavena, P., and Rescigno, M (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature Immunol. 6:507-514.

Johansson-Lindbom, B., Svensson, M., Pabst, O., Palmqvist, C., Marquez, G., Forster, R., and Agace, W.W. (2005) Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing. J.Exp. Med., 202:1063-1073.

Huang, F.P., Platt, N., Wykes, M., Major, J.R., Powell, T.J., Jenkins, C.D. and MacPherson, G.G. (2000) A discrete population of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J.Exp. Med. 191:432-444.

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Watanabe, T., Kitani, A., Murray, P.J. and Strober,W. (2004) NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper I responses. Nat. Immunol. 5:800-808.


Definition of the factors (immune a dietary) that influence dendritic cell maturation/function in the mucosal environment, including factors derived from epithelial cells and the commensal microflora.

Elucidation of the chemokines that control the movement of dendritic cells into mucosal sites and around mucosal sites.

Definition of the factors that influence production of factors that differentially influence effector cell and regulatory cell development including the cell-cell interactions involved.


Construction of mice that lack dendritic cell-specific surface molecule and intra-cellular molecules that control recognition of microbial components (TLR ligands/antigens) and are involved in essentially signaling functions that regulate dendritic cell maturation and activation.

Examination of the function of dendritic cells in murine models of inflammation with relation to changes in the sub-populations of dendritic cells present in the inflamed mucosa and the functions of these dendritic cells. Creation of mouse models characterized by dendritic cell dysfunction that leads to mucosal inflammation.

Elucidation of the function of TLR and NLR microbial-recognition molecules as these relate to positive and negative dendritic cell responses. This includes the propensity of these molecules to induce inhibitory factors that down-regulate responses.

Long -Term Goals (8-10 years)

In-depth elucidation of the intra-cellular signaling pathways that define TLR, NLR, CD40, IL-1b, TNF, and other relevant signals that act on dendritic cells and determine the unique behavior of mucosal dendritic cells.

Definition of the effects of co-ordinate signaling on the overall response of mucosal dendritic cells.

Creation of mouse models characterized by dendritic cell dysfunction that lead to mucosal inflammation. Comparison of these mice with the function of dendritic cells derived from normal humans and those with IBD.

Research Advance #4The traffic of mucosal cells to various parts of the mucosal immune system

The last decade has seen major advances in the understanding of how and why the mucosal immune system is unified by a cell circulation system that ensures that cells generated with the inductive areas of the system, the Peyer’s patche, and other lymphoid follicles, “home” back to the effector areas of the system, the GI lamina propria and other “diffuse” mucosal areas in other organs. Older studies focused on the role of integrin/integrin receptors in this process, particularly the role of the 47/MAdCAM-1 combination in gut home. However, in newer work it has been established that regional expression of epithelial chemokines in the small and large intestine play an indispensable part in the homing process: IgA plasma cell migration to the small intestine requires the CCL25/CCR9 (ligand/receptor) pair and the CCL28/CCR10 pair for migration to the colon and other mucosal tissues. In addition, the CCL25/CCR9 pair governs the corresponding migration of gut homing T cells to the small intestine; however, in this

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case, the chemokine pair guiding migration of these cells to the large intestine is still not defined. A major new finding is that retinoic acid (vitamin A) acting through the retinoic acid receptor (RAR) induces IgA plasmablasts and T cells to express homing receptors for the gut. This finding complements the older finding that TGF- induces the expression of the E7 integrin by completing our knowledge of the inducer of B cell gut homing receptors. Finally, the role of chemokine ligand-receptor interactions governs other aspects of mucosal immune function, including the exit of T cells and dendritic cells from the mucosal tissues into draining lymph nodes, the entry of CD8+ thymic emigrants into the mucosal to become intra-epithelial lymphocytes, and the movement of dendritic cells to sub-epithelial locations. It is now apparent that the traffic of cells in, around, and out of the mucosal immune system is a highly choreographed series of events that depends in great measure on chemokine ligands and their interaction with chemokine receptors.

Citations:

Svensson, M., Marsal, J., Ericsson, A., Carramolino, Broden, T., Marquez, G., and Agace, W.W. (2002) CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa. J. Clin. Invest. 110:1113.

Kunkel, E.J., Kim, C.H., Lazarus, N.H., Vierra, M.A., Soler, D., Bowman, E.P., and Butcher, E.C. (2003) CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J. Clin. Invest. 111:1001.

Hieshima, K., Kawasaki, Y., Hanamoto, H., Nakayama, T., Nagakubo, D., Kanmaru, A., and Yoshie, O. (2004) CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells. J. Immunol. 173: 3668.

Iwata, M., Hirakiyama, A., Eshima, Y., Kagechika, H., Kato, C., and Song, S.Y. (2004) Retinoic acid imprints gut-homing specificity on T cells. Immunity 21: 527.

Debes, G.F., Arnold, C.N., Young, A.J., Krautwald, S., Lipp, M., Hay, J.B., and Butcher, E.C. (2005) Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nat. Immunol. 6:889.


Definition of chemokine-chemokine receptor interactions that are critical for the entry of T cells into the large bowel and the entry of CD8+ thymic emigrants into the mucosa to become intra-epithelial lymphocytes. Likewise, definition of the interactions necessary for the entry of cells into other mucosal organs such as stomach, live, and oral cavity.

Definition of the chemokine-chemokine receptor interactions or other cell-cell interactions that contribute to the retention of lymphocytes and other cells in mucosal tissue.

Elucidation of the migration of various classes of regulatory T cells into and out of mucosal tissues.

Exploration of the molecular signaling and gene activation pathways that are involved in retinoic acid-ROR signaling giving rise to mucosal homing receptors.


Continued exploration of the value of blocking gut homing receptors in the prevention/amelioration of IBD and GvHD. These studies are a continuation of extensive studies already performed or underway of antibodies or other agents that impede the activity of the integrin homing receptor, 47

Exploration of the homing properties of cells differ in the face of inflammation.

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Construction of tissue-specific knock-out mice that lack key components of the gut homing apparatus and thus manifest various kinds of mucosal immune abnormalities.


Development of systems approach to the study of lymphocyte and dendritic cell homing that integrates the many factors that affect this process. This approach would allow prediction of cell homing based on the combined effects on integrin/chemokine effects with the effects of other influences such as the state of cell differentiation, the presence of various cytokine, and the influence of Toll-like receptors on homing.

Research Advance #5Mucosal unresponsiveness (oral tolerance) and mucosal regulatory T cell development

It has long been known that oral administration of a protein antigen leads to a subsequent state of unresponsiveness to that antigen as a result of a phenomenon known as oral tolerance induction. Oral tolerance is also manifest in the lack of an overt inflammatory response to the mucosal microflora, despite the proximity and persistence of this source of potential antigens. The last decade has witnessed a great advance in the understanding of this phenomenon and the possible harnessing of its underlying mechanisms to the therapy of GI inflammation. A significant step forward came with the demonstration that while oral tolerance could be due to exposure of mucosal cells to high doses of antigen (in the absence of adequate T cell co-stimulation), it is more characteristically due to exposure of mucosal cells to low doses of antigen and the induction of regulatory T cells. Further work has established that the most important type of regulatory cell mediating oral tolerance is the so-called “natural” regulatory T cells that are defined by their expression of surface markers such CD25, CTLA-4, and CD103, and GITR and, more importantly, by their expression of a particular transcription factor known at foxP3. While still controversial, it is likely that natural regulatory T cells cause inhibition of responses because they express surface TGF- and secrete TGF- and bring this suppressive cytokine to bear on antigen-presenting cells. For the most part, these cells develop in the thymus (under the influence of IL-2) and bear antigen receptors recognizing self-antigens; however, it is likely that the also bear receptors recognizing antigens associated with the gut microflora because these antigens have access to the circulation and can enter the thymus. Thus, one scenario for the high prevalence of regulatory cells in the mucosal is that such cells develop in the thymus and then migrate to the mucosa where they re-encounter microflora antigens and undergo clonal expansion. A second possible reason for their high prevalence in the gut is that natural suppressor T cells can be induced to expand in peripheral tissues by TGF-, a cytokine that is produced by epithelial cells and thus is present at a high steady-state level in the gut tissue. Another type of regulatory cell that can develop in the mucosa and that may also mediate oral tolerance is the so called Tr1 regulatory cell. In contrast to the natural regulatory cell, the Tr1 cell lacks high level foxp3 expression and develops in relation to exogenous rather than self antigens; thus, it may be induced by protein antigen feeding or by infection of the GI tract. This regulatory T cell is induced by IL-10 and other cytokines and produces high amounts of IL-10. The factors that determine whether a given mucosal antigenic stimulus will result in (positive) immune effector response important for host defense or a (negative) regulatory T cell response important for maintenance of an unresponsive state and prevention of mucosal inflammation are still poorly understood. One working hypothesis is that the regulatory response is a default response that occurs in the absence of a strong innate immune response driven by TLR ligands. The latter tends to drive high IL-12/IL-23 responses that over-ride and even inhibit regulatory responses. The importance of the regulatory response has become apparent in the study of murine models of inflammation in which it was shown in a number of models that lack of regulatory cell generation leads to colonic inflammation. In one model, the cell transfer model, it has been shown that transfer of cell populations lacking regulatory T cells to RAG-2 KO mice leads to colonic inflammation, whereas cell populations containing regulatory T cells remain free of colonic inflammation. In this system, the regulatory cells seem to require TGF-, since they do not prevent inflammation in recipients that lack TGF- receptors. In another model, the hapten induced model of TNBS-colitis, oral feeding of

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TNP-substituted protein prevents colitis and this prevention is also TGF- mediated. This model ties the regulation of experimental colitis to oral tolerance. Finally, using the cell transfer model, it has been shown that provision of regulatory T cells to mice with established colitis, abrogates the colitis. This finding raises the question of whether human IBD is caused by lack of sufficient regulatory T cell activity and whether IBD can be treated by enhancing such activity. A possible example of the latter is the resetting of mechanisms that ordinarily down-regulate suppressor cytokines such as the down-regulation of TGF- function by Smad7.

Citations:

Weiner, H.L. (2000) Oral tolerance, an active immunologic process mediated by multiple mechanisms. J. Clin. Invest.106:935-957.

Nakamura, K., Kitani, A., Fuss, I., Pedersen, A., Harada, N. and Strober, W. (2004) TGF-beta 1 plays an important role in the mechanism of CD4+CD23+ regulatory T cell activity in both humans and mice. J. Immunol. 172:834-842.

Fuss, I.J., Boirivant, M., Lacy, B., and Strober, W. (2002) The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900-908.

Fahlen L., Read, S., Gorelik, L., Hurst, S.D., Coffman, R.L., Flavell, R.A., and Powrie, F. (2005) T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. J. Exp. Med. 201:737-746.

Mottet C., Uhlig, H.H., and Powrie, F. (2003) Cutting Edge: Cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170:3939-3943.

Zheng, S.G., Grey, J.D., Ohtsuka, K., Yamagiwa, S., and Horwitz, D.A. (2002) Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25+ precursors. J. Immunol. 169:4183-4189.


Elucidation of the innate immune responses that determine whether a mucosal immune response will result in an effector cell response characterized by inflammation or a regulatory cell response characterized by tolerance.

Discovery of the dendritic cell interactions that result in the generation of regulatory T cells and the relation of these interactions to microflora antigens and TLR ligands on the one hand and nominal (dietary) antigens on the other.

Further exploration of the nature of the immunological milieu of the mucosa enhancing or retarding the development of regulatory T cells. Acquisition of definitive data on the production of cytokines by epithelial cells that either enhance or retard regulatory T cell development.

Discovery of markers for the identification of regulatory T cells in the mucosal tissues and the measurement of the number and function of regulatory T cells in IBD tissues.

Define differences between inducible regulatory cells and regulatory cells that develop naturally with respect to cell markers, mode of suppression, and persistence in the host.

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Elucidation of methods of generating regulatory cells ex vivo for administration and treatment of patients with IBD.

Exploration of the signaling pathways involved in the generation of regulatory T cells via TGF- or the inhibition of such generation by IL-6.

Achieve a better understanding of the biology of regulatory T cells with relation to the function of foxp3 and other intra-cellular factors that control regulatory cell function.

Define the nature of regulatory cells in humans with regard to the existence of unique human regulatory cell markers and functions.


Elucidation of the genetic basis of regulatory T cell function. Identification of genes that affect how regulatory T cells are generated in the periphery and how regulatory T cell function is maintained and inhibited.

Development of gene therapy approaches to the enhancement of regulatory T cell function in the treatment of chronic inflammatory states.

Research Advance #6Effector T cell responses in the GI tract and the pathogenesis of IBD

There is general agreement that chronic inflammation of the GI tract (both ulcerative colitis and Crohn’s disease) represents an abnormal immunologic response to antigens as well as to TLR ligands in the mucosal microflora. This abnormal response can arise from an excessive effector T cell response to such antigens or as a normal effector T cell response to such antigens that is not sufficiently restrained by regulatory T cells. Compelling evidence for this formulation comes from the extensive study of murine models of mucosal inflammation over the last decade that shows that experimental inflammation arising from diverse causes does not occur if the mice are maintained in a germ-free environment. Recently additional evidence favoring this view has come from studies of the basis of inflammation in 15% of Crohn’s disease patients who bear mutation in the CARD 15 gene that encodes NOD2, an intra-cellular member of the NLR family of proteins that recognizes and responds to a peptide, muramyl dipeptide (MDP), derived from the peptidoglycan component of the bacterial cell wall. In these studies it was shown that while NOD2 stimulated by MDP has a modest ability to activate NF-B and induce cytokine synthesis, its major function is that of down-regulation of TLR responses, particularly those induced by its parent molecule, peptidoglycan, acting through the TLR2 receptor. Thus, these studies support the view that Crohn’s disease susceptibility arising from CARD 15 mutations is due to faulty down-regulation of TLR responses leading to excessive responses to antigens associated with the microflora. One additional and important point is that CARD 15 mutations may be a necessary but not a sufficient factor in the pathogenesis of Crohn’s disease. This view arises from studies showing that mice with NOD2 deficiency do not develop gut inflammation unless they are exposed to microbial antigen and have resident T cells that can respond to that antigen. This suggests a “two hit” theory of Crohn’s disease pathogenesis: a first hit involving a dysregulated innate immune response represented in this case by NOD2 deficiency and a second hit involving the presence of effector T cells that can respond to one or another antigen in the microbial microflora. This theory is currently supported by work showing that in both human and murine systems, adaptive responses to flagellin antigen seems to be a characteristic of some patients with Crohn’s disease. Another factor leading to excessive responses to microflora antigens that is likely to be involved in the pathogenesis of disease involves the barrier function of the gut epithelium. There is now some evidence that while the bacterial microflora in IBD may not in itself be qualitatively abnormal, the microflora has an abnormal relation to the mucosal immune system such that

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the latter becomes over-stimulated and inflammation results. Evidence for this comes from the fact the recent discovery that defective production of -defensins is associated with Crohn’s disease and that this abnormality is aggravated in patients with one type of CARD 15 mutation, possibly due to the fact that NOD2 is also expressed in Paneth cells and may regulate the production of -defensins in such cells. In addition, mice with defective epithelial barrier function are susceptible to experimental colitis and certain of identified genes associated with Crohn’s disease, notably the OCT/N gene, is thought to affect epithelial cell function. Whether these epithelial cells defects are sufficient to cause disease or require a concomitant abnormal of the mucosal immune system remains to be seen.

The fundamental defects resulting in IBD, result from re-enforcing genetically determined defects in immune function (or epithelial cell function) some of which are described above or others that are as yet poorly understood. Regardless of this verisimilitude, the immunologic effector cell response mediating the inflammation is channeled through a common final pathway, one specific for Crohn’s disease and the other for ulcerative colitis. The effector cell response in Crohn’s disease was presaged by studies of mouse models of colitis resembling Crohn’s disease that were universally shown to respond to treatment with antibody to IL-12 (anti-IL-12p40). This, plus the fact that the cells in the lesions produced high amounts of IFN-g but little or no IL-4 seemed to indicate that the inflammation was a Th1-mediated effector cell process. Ultimately, this conclusion was reinforced by a clinical study of the efficacy of anti-IL-12p40 in patients with Crohn’s disease that showed that most patients were remarkably responsive to this therapy. However, recently evidence has accumulated that in the cell transfer model of colitis mentioned above, most of the inflammation can be attributed to IL-23 rather than IL-12 and that the IL-23 was acting through its capacity to maintain and support the differentiation of cells producing IL-17 (Th17 cells). In the light of this finding, it was felt that the efficacy of anti-IL-12p40 was due to the fact that the p40 chain is shared by IL-12 and IL-23 and thus the antibody was mainly blocking IL-23 rather than IL-12 in previous mouse and human studies of this antibody. This seemed to be supported by the observation that anti-IL23p19, an antibody specific for IL-23, was as effective as anti-IL-12p40 in treating cell transfer colitis. One caveat to these new data is that in another model of colitis, the hapten-induced TNBS-colitis, absence of IL-23 actually resulted in more severe disease; in this model, there is evidence that IL-12p40 is indeed the major driving cytokine and that role of IL-23, if any, is to down-regulate the IL-12p40 response. Studies conducted in humans show that both IL-12 and IL-23 (as well as IFN- and IL-17) are elevated in lesional tissue, suggesting that both the Th1 and Th17 systems are operative in the human disease. The inflammation in the second major form of IBD, ulcerative colitis, is clearly not a Th1- or Th17-mediated process since in this disease neither IL-12 nor IL-23 is elevated. Recently, it has been shown that this disease resembles a second hapten-induced experimental colitis, namely oxazolone colitis, in that increased numbers of NKT cells are found in lesions and these cells produce increased amounts of IL-13. Furthermore, it has been shown that IL-13 can mediate tissue injury by acting as an autocrine factor that induces NKT cells to manifest increased cytotoxic activity toward epithelial cells and by acting directly on epithelial cells to increase permeability and apoptosis. On the basis of these data, one working hypothesis on the pathogenesis of ulcerative colitis is that it is a Th2-like process mediated by NKT cells producing IL-13.

Citations:

Bouma, G., Strober, W. (2003) The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 3: 521-533.

Ogura, Y., Bonen, D.K., Inohara, N., Nicolae, D.L., Chen, F.F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R.H., Achkar, J.P., Brant, S.R., Bayless, T.M., Kirschner, B.S., Hanauer, S.B., Nunez, G., and Cho, J.H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411: 603-606.

Watanabe, T., Kitani, A., Murray, P.J., Wakatsuki, Y., Fuss, I.J., and Strober, W. (2006) Nucleotide binding oligomerization domain 2 deficiency leads to dysregulated TLR2 signaling and induction of antigen-specific colitis. Immunity 25: 473-485.

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Wehkamp, J., Salzman, N.YH., Porter, E., Nuding, S., Weichenthal, M., Petras, R.E., Shen, B., Schaeffeler, E., Schwab, M., Linzmeier, R., Feathers, R.W., Chu, H., Lima, H., Jr., Fellermann, K., Ganz, T., Stange, E.F., and Bevins, C.L. (2005) Reduced Paneth cell alpha-defensins in ileal Crohn’s disease. Proc Natl Acad Sci USA 102: 18129-18134.

Mannon, P.J., Fuss, I.J., Mayer, L., Elson, C.O., Sandborn, W.J., Present, D., Dolin, B., Goodman, N., Groden, C., Hornung, R.L., Quezado, M., Yang, Z., Neurath, M.F., Salfeld, J., Veldman, G.M., Schwertschlag, U., and Strober, W. (2004) Anti-interleukin-12 antibody for active Crohn’s disease. N Engl J Med 351: 2069-2079.

Hue, S., Ahern, P., Buonocore, S., Kullberg, M.C., Cua, D.J., McKenzie, B.S., Powrie, F., and Maloy, K.J. (2006) Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med 203: 2473-2483.

Fuss, I.J., Heller, F., Boirivant, M., Leon, F., Yoshida, M., Fichtner-Feigl, S., Yang, Z., Exley, M., Kitani, A., Blumberg, R.S., Mannon, P., and Strober, W. (2004) Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. 113: 1490-1497.


Elucidate the function of NOD2 in murine systems with the use of NOD2 KO and KI mice as well as NOD2 transgenic mice with cell-specific over-expression. In particular, define the mechanism of NOD2 inhibition of TLR responses.

Create murine knock-out models of other genes so-far identified as susceptibility genes in IBD to determine the role of these genes in mucosal immune responses and/or epithelial barrier function.

Explore the function of novel genes that affect mucosal responses to the gut microflora, particularly those genes that have a direct or indirect impact on epithelial barrier function.

Create new models of mucosal inflammation that more closely mimic human Crohn’s disease and ulcerative colitis to determine the role of IL-12 and IL-23 in Crohn’s disease and IL-13 in ulcerative colitis.


Create murine knock-out models of genes identified as susceptibility genes in IBD to determine the role of these genes in mucosal immune responses and/or epithelial barrier function.

Identify genetically determined hyper-responsiveness to candidate antigens in the mucosal microflora. Create murine models to verify that such hyper-responsiveness can lead to mucosal inflammation.

Evaluate the efficacy of novel agents that affect the final common pathways of mucosal inflammation in inflammatory bowel disease, including agents that affect the traffic of cell in the mucosal immune system, key effector cytokines such as IL-23p19, IL-17, IL-22, and IL-13. Complete on-going studies of IL-12p40 to determine if this can be a viable alternative for the treatment of Crohn’s disease.

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Create mice with multiple gene defects simulating the defects found in patients to determine if such mice develop inflammatory bowel disease under appropriate environmental conditions.

Evaluate the treatment of patients with Crohn’s disease (or ulcerative colitis) with identified gene defects using stem cells repleted with normal genes.

Research Advance #7The IgA response and mucosal vaccination

Undoubtedly the most unique aspect of the mucosal immune system is that the B cell response characteristic of this system is an IgA B cell response. The reason for this mucosal isotype skewing remains unclear; however, since class-switch recombination (CSR) resulting in IgA B cells has an absolute requirement for TGF- and since TGF- is particularly associated with cells present in the mucosal environment, a working hypothesis is that mucosal IgA dominance is a direct result of the availability of this cytokine in mucosal tissues. In an older view, IgA B cells were thought to arise exclusively in mucosal follicles present mainly in the Peyer’s patches or in isolated intestinal lymphoid nodules scattered through the intestine, and indeed it could be shown that these follicles were the only ones containing substantial numbers of developing IgA B cells. There was substantial evidence that IgA B cell stimulation at these sites was initiated by B cell receptor (BCR)-mediated stimulation by protein antigens entering the follicle and required cognate interactions between T cells and conventional (B2) B cells, including interactions involving CD40L and CD40. Following such stimulation, the B cells were imprinted to migrate through the draining nodes and the lymph system back to the “diffuse” mucosal lymphoid areas in the lamina propria of mucosa. It is possible, but still unproven, that such cognate T cell-B cell interactions resulting in IgA B cells requires a T cell bearing surface TGF- or secreting TGF- as do regulatory T cells. On this basis, the regulatory T cell may be uniquely involved in IgA B cell development.

In recent years, this follicle and T cell-centered view of IgA B cell differentiation has had to make room for a second pathway of IgA B cell development since it is clear that IgA B cells develop in relation to exposure to components of the commensal microflora in the absence of T cells or CD40L or, indeed of mucosal follicles. This pathway of IgA B cell development could be viewed as a more “innate” pathway given evidence that it occurs in response to innate receptors such as TLR receptors and may be triggered by T cell-independent non-protein antigens. In addition, this pathway may utilize, at least in part, an unconventional B cell, the B1 B cell, that follows an independent line of development from conventional B cells. It remains unclear where this type of IgA B cell actually undergoes IgA-specific CSR since initial reports that this occurs in the lamina propria have recently been refuted. On the other hand, there is a very recent report that the CSR occurs in close juxtaposition to epithelial cells (in epithelial pockets) and that the switching in these areas occurs when the epithelial cells are stimulated by TLR ligands and secrete TSLP (mentioned above in relation to epithelial effects on dendritic cells) as well as BAFF, a factor that may induce CSR as well as terminal B cell differentiation. The TGF- necessary for IgA-specific CSR could be coming from epithelial cells or from nearby dendritic cells.

The fact that a single cytokine, TGF-, is at once involved in the induction of regulatory cell responses and in the induction of IgA responses is probably not fortuitous, since the mucosal immune system, as already alluded to above, is oriented toward regulation of the commensal organisms in the gut lumen. Indeed, the IgA produced by the T cell-independent process discussed above may have a particular role in controlling the entry of antigens derived from commensal organisms into the area beneath the epithelium since such antigens would be susceptible to interception by IgA and removal from the system via polyimmunoglobulin receptor transport across the epithelium. Thus, if we turn our attention to the induction of IgA antibody responses to protein components of potential pathogens via the administration of oral or intra-nasal (or intra-rectal) vaccines, we may be dealing mainly with the IgA responses occurring in the follicles via cognate T cell interactions.

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In the latter context, it has been known for some time that such responses require the use of mucosal adjuvants, i.e., substance that when administered via the mucosal immune system (i.e., orally or intra-nasally) have properties that allow them to stimulate the production of factors that promote IgA-specific CSR and the expansion of IgA B cells resulting from the switch, rather than the default response discussed above that gives rise to oral tolerance. Extensive study of one particularly potent mucosal adjuvant, cholera toxin, has provided important insights into the way mucosal adjuvants function. Cholera toxin (the holotoxin) consists of two chains, an A chain that has potent ADP ribosyltransferase activity and thus stimulates cells via a G protein-mediated activation process. The second or B chain is the anchor chain because of this ability to bind to cell surface GM1-ganglioside. The holotoxin cannot be used as an adjuvant because its binding to epithelial cells leads to GI fluid loss and diarrhea. Recently, this problem has been overcome by fusing the A chain to staphylococcus protein A to form a protein (CTA-1DD) that binds avidly to B cells and other antigen-presenting cells but does not cause GI symptoms. Studies of the mechanism of action of CTA-1DD show antigen bound to CTA-1DD binds to marginal zone dendritic cells that then migrate to the T cell zone and express the co-stimulatory molecules, CD86. This induces the movement of antigen-specific CD4+ T cells into B cell follicles and subsequent germinal center formation. CTA-1DD lacking ribosylation capability does not have this effect and is a poor adjuvant and while CTB binds to dendritic cells, it does not induce their migration or maturation; this may explain why CTB given orally is actually an inducer of oral tolerance rather than immunization. Thus, the picture that emerges is that mucosal adjuvants induce mucosal immunization rather than tolerance because they activate dendritic cells to express surface molecules and cytokines that activate effector T cells rather than regulatory cells. These fruitful studies of cholera toxin-based mucosal adjuvants provide a template for the study of other mucosal adjuvants, including those that address all-important mucosal T cell responses.

A very practical aspect of pursuing increased knowledge concerning in the induction of IgA and other mucosal responses arises from the fact that the mucosal system is somewhat separate from the “systemic” immune system by virtue of the homing receptors that mandate the traffic of cells originating in the inductive sites of the system to effector sites. This carries the important implication that only mucosal immunization can effectively deal with pathogenic invasion of the mucosa. This point is particularly relevant to the prevention of HIV disease given recent evidence that the gastrointestinal tract is a major site of initial HIV development and a major reservoir of established HIV infection.

Citations:

Strober, W., Fagarasan, S., and Lycke, N., (2005) IgA B cell development. In: Mucosal Immunology, 3rd Edition. J. Mestecky, M.E. Lamm, W. Strober, J. Bienenstock, J.R. McGhee and L. Mayer, eds. Academic Press, Boston, pp. 583-616.

MacPherson, A.J., Gatto, E., Sainsbury, E., Harriman, G.R., Hengartner, H., and Zinkernagel, R.M. (2000) A primitive T cell-independent mechanism of intestinal IgA responses to commensal bacteria. Science 288:2222-2226.

Fagarasan, S., Muramatsu, M., Suzuki, K., Nagaoka, H., Hiai, H., and Honjo, T. (2002) Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298:1424-1427.

Bergqvist, P., Gardby, E., Stensson, A., Bemark, M., and Lycke, L. (2006) Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers. J. Immunol. 177:7772-7783.

Grdic, D., Ekman, L., Schon, K., Lindgren, K., Mattsson, J., Magnusson, K.E., Ricciardi-Castignoli, P., and Lycke, N. (2005) Splenic marginal zone dendritic cells mediate cholera toxin adjuvant effect: dependence on ADP ribosylation activity of the holotoxin. J. Immunol. 175:5192-5202.

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Xu, W.,Bing, H., Chiu, A.,Chadburn, A., Shan, M., Buldys, M., Ding, A., Knowles, D.M., Santini, P.A., and Cerutti, A. (2007) Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nat. Immunol. 8:294-303.

Holmgren, J., and Czerkinsky, C. (2005) Mucosal Immunity and Vaccines. Nature Medicine, supplement.


Elucidation of the full range of epithelial or stromal cell factors/cytokines that are involved in the elaboration of IgA B cells. Determination of whether these factors/cytokines operate at the level of class switch or terminal differentiation.

Delineation of the extent of T cell-independent IgA produced in the mucosa and its role in maintaining mucosal barrier function and/or antigen sampling. Delineation of the regulation of commensal organisms in the absence of IgA production.

Clarification of the types of T cells involved in cognate interactions leading to IgA differentiation and regulatory T cells.

Development of anti-immunization agents administered orally that induce tolerization rather than immunization such as cholera toxin B chain.


Accumulation of more specific knowledge of the mechanism of action of various mucosal adjuvants at the level of dendritic cells and T cells.

Development of new adjuvants that target particular aspects of the mucosal immune response: TLR ligands alone or bound to cholera toxin adjuvants; ISCOMs for the targeting of antigen to dendritic cells; and, various cytokines, such as IL-12, IL-23, IL-15, etc.

Development of optimized vaccine schedules for particular infectious agents including intra-nasal and intra-rectal vaccine administration.


Development of effective vaccines that induce IgA and/or T cell responses via mucosal immunization; this includes the development of mucosal vaccines for many of the major epidemic viral infections and for HIV disease.

Development of mucosal vaccines that induce high level “innate” IgA responses addressing the entry of the intestinal microflora into the mucosa for use in inflammatory bowel disease due to defects of barrier function.

Goals and Challenges/Steps to Overcome Challenges:

1. Animal Resources: The Challenge: Animal models are increasingly necessary for the proving the creditability of hypotheses in the real world. At the same time, animal models have become increasingly complex since they require the introduction of gene defects in specific tissues/cells (sometimes at specific time points). While such models are theoretically attainable, they are expensive and time consuming to develop.Steps To Overcome Challenge: Centralized animal resources will need to be developed as a repository of potentially useful and currently useful models for the general research community. This will include the capability of preserving animal models as frozen embryos or sperm or the long-term

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maintenance of experimental models in centrally supported facilities. In addition, it will involve the provision of resources for the development of new animal models by groups that can create the molecular constructs and perform the embryonic manipulations that are necessary for creation of such models.

2. Germ-Free Facilities: The Challenge: A great number of the research initiatives suggested above involve the study of live experimental animals under conditions in which the commensal microflora of the gut is strictly defined and controlled.

Steps To Overcome Challenge: Centralized germ-free facilities are necessary to provide qualified researchers with germ-free and/or microflora-defined mice for study under various conditions. An important need of such facilities is the development of the capability of sending these mice to distant locations so that research of this sort can theoretically be conducted anywhere.

3. Cells: The Challenge: Cells derived from animal models with defined attributes are necessary for in vitro studies. In some cases these are cells that are not presently sustainable in culture for long periods of time. Use of cell lines from animal models mentioned above, or humans with diseases as mentioned below, are essential for characterization of signaling pathways and secretory potential of various types of cells.

Steps To Overcome Challenge: This challenge has developmental and logistical components. The developmental component involves the acquisition of techniques for the long-term maintenance of cells in culture under circumstances in which the cells do not undergo major changes in characteristics. This developmental component applies, at a minimum, to the provision of native epithelial cell lines and dendritic cell lines. The logistical component applies to the development of central facilities to both acquire and maintain these cells for distribution to qualified investigators.

4. Primate Studies: The Challenge: Many studies in mucosal immunity, particularly those focusing on vaccine development, ultimately require the vetting in primates. The care and maintenance of primates for studies requires a cadre of professions with direct and extensive experience in this endeavor.

Steps To Overcome Challenge: Creation of primate facilities with in-house professional staff to execute complex studies in primates.

5. Genetic Research: The Challenge: To an increasing extent, progress in mucosal immunity, especially as it relates to the elucidation of mucosal disease, requires the ability to identify genes and gene function. While considerable intra-structure has been constructed for the identification of disease genes in disease, often relying on the identification of statistical associations between disease occurrence and SNP polymorphisms, there is little effort to translate these genetic findings into pathophysiologic insights into disease pathogenesis.

Steps To Overcome Challenge: Better integration of genetic research with research oriented to understanding the function of newly discovered genes of interest. Dedicated support of research that is poised to take up the challenge of translating genetic discoveries into pathophysiological insights.

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Patient Profile Topic

Inflammatory bowel disease (ulcerative colitis and Crohn’s disease) is the major medical problem addressed in this research sub-section. A challenge to research in this area is the acquisition of materials from large numbers of patients for genetic and/or immunologic studies. For genetic studies it is going to be increasingly important for qualified researchers to have access to patient DNA samples that are linked to extensive clinical and epidemiologic information. Appropriate controls relating to the dissemination of such materials must be in place that do not, at the same time, render their access too obstructive and burdensome. For immunologic studies, core components are necessary for the isolation and storage of live and preserved tissues and cells in both a purified and whole tissue form. In certain cases, new techniques for the preservation and storage of tissue and cells have to be devised.

Graphics and Images None

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Documents

Working Group #01 Consolidated Post-Conference Call Report.doc